Attenuated Mycobacterium tuberculosis vaccines

ABSTRACT

Non-naturally occurring mycobacteria in the  Mycobacterium tuberculosis  complex are provided. These mycobacteria have a deletion of an RD1 region or a region controlling production of a vitamin, and exhibit attenuated virulence in a mammal when compared to the mycobacteria without the deletion. Also provided are non-naturally occurring mycobacteria that have a deletion of a region controlling production of lysine, and mycobacteria comprising two attenuating deletions. Vaccines comprising these mycobacteria are also provided, as are methods of protecting mammals from virulent mycobacteria using the vaccines. Also provided are methods of preparing these vaccines which include the step of deleting an RD1 region or a region controlling production of a vitamin from a mycobacterium in the  M. tuberculosis  complex.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/358,152, filed Feb. 19, 2002. That application is incorporated byreference herewith in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The present invention was supported by NIH Grant No. AI26170. As such,the U.S. Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention generally relates to live bacterial vaccines. Morespecifically, the invention is related to novel Mycobacterium sp.compositions, and the use of those compositions to protect mammalsagainst disease caused by virulent Mycobacterium sp.

(2) Description of the Related Art

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U.S. Pat. No. 6,271,034.

U.S. Pat. No. 5,504,005.

There exists an urgent need for a novel tuberculosis (TB) vaccine asthere are more than 8 million new cases of tuberculosis and more than 2million deaths reported each year by the WHO (Dye et al., 1999). Thediscovery of the causative agent of TB, Mycobacterium tuberculosis, byRobert Koch in 1882 opened up the possibility for a novel vaccine (Koch,1882). Since then, numerous attempts to develop attenuated vaccinesagainst tuberculosis have failed, including tuberculin (a proteinextract of killed tubercle bacilli) developed by Dr. Koch himself. Thisfailure of tuberculin to protect led to a “firm conviction that immunitycould only be established by inducing a definite, albeit limited,tuberculosis process” Grange et al., 1983). Thus, numerous labs set outto follow the example of Dr. Louis Pasteur for viruses and enrichattenuated mutants of the tubercle bacillus following repeatedpassaging.

In order to test the hypothesis that a tubercle bacillus isolated fromcattle (now known as M. bovis) could transmit pulmonary tuberculosisfollowing oral administration, Drs. Calmette and Guerin developed amedium containing beef bile that enabled the preparation of finehomogenous bacillary suspensions (Calmette and Guerin, 1905). An M.bovis strain obtained from Dr. Norcard, was passaged every 21 days inthis medium and after the 39^(th) passage, the strain was found to beunable to kill experimental animal (Gheorghiu, 1996). “Between 1908 and1921, the strain showed no reversion to virulence after 230 passages onbile potato medium” (Id.), which is consistent with the attenuatingmutation being a deletion mutation. In the animal studies that followed,the strain (‘BCG’) was found to be attenuated but it also protectedanimals receiving a lethal challenge of virulent tubercle bacilli(Calmette and Guerin, 1920). BCG was first used as a vaccine againsttuberculosis in 1921. From 1921 to 1927, BCG was shown to haveprotective efficacy against TB in a study on children (Weill-Halle andTurpin, 1925; Calmette and Plotz, 1929) and adopted by the League ofNations in 1928 for widespread use in the prevention of tuberculosis. Bythe 1950's after a series of clinical trials, the WHO was encouragingwidespread use of BCG vaccine throughout the world (Fine and Rodrigues,1990). Although an estimated 3 billion doses have been used to vaccinatethe human population against tuberculosis, the mechanism that causesBCG's attenuation remains unknown.

Mahairas et al. (1996) first compared the genomic sequences of BCG andM. bovis using subtractive hybridization and found that there were threemajor deletions (named RD1, RD2, and RD3) present in the genome of M.bovis, but missing in BCG. Behr et al. (1999) and others (Gordon et al.,2001) later identified 16 large deletions, including RD1 to RD3, presentin the BCG genome but absent in M. tuberculosis. These authors concludedthat 11 of these 16 deletions were unique to M. bovis, while theremaining 5 deletions were unique to BCG. They also found that one ofthese 5 deletions, designated RD1 (9454 bp), is present in all of theBCG substrains currently used as TB vaccines worldwide and concludedthat the deletion of RD1 appeared to have occurred very early during thedevelopment of BCG, probably prior to 1921 (Behr et al., 1999).

The development of insertional mutagenesis systems for BCG and M.tuberculosis (Kalpana et al., 1991), transposon mutagenesis systems(Cirillo et al., 1991; McAdam et al., 1995; Bardarov et al., 1997) andallelic exchange systems (Balasubramanian et al., 1996; Pelicic et al.,1997) led to the isolation of the first auxotrophic (nutrient-requiring)mutants of these slow-growing mycobacteria. Auxotrophic mutants of BCGand M. tuberculosis have been shown to confer protection to M.tuberculosis challenges with variable efficacies (Guleria et al., 1996;Smith et al., 2001). However, a head-to-head comparison of BCG to aleucine auxotroph of BCG showed that a single immunization elicited nopositive skin-test and imparted little immunity to challenges with M.tuberculosis or M. bovis (Chambers et al., 2000). In contrast, amethionine auxotroph of BCG that grows in vivo did confer significantprotection to challenge to both M. tuberculosis and M. bovis (Id.). Asingle dose of a leucine auxotroph of M. tuberculosis failed to elicitprotection as good as BCG in BALB/c mice (Hondalus et al., 2000). Theseresults suggest that optimal immunity against M. tuberculosis requiressome growth of the immunizing strain. Double mutants of M. tuberculosishave also been created (Parish and Stoker, 2000), but whether suchmutants are improved over single attenuating mutants in protectingmammals against challenge with a virulent mycobacterium, particularlywhen the host is immunocompromised, has not been established.

It is also worth noting that in the study of Chambers et al. (2000),both BCG and the BCG mutants seemed to protect better against M. bovischallenge than M. tuberculosis. If we assume the reverse correlate istrue, we could hypothesize that optimal immunity against M. tuberculosiscould be achieved with M. tuberculosis-derived mutant that grew in themammalian host.

Based on the above, there remains a need for improved live mycobacterialvaccines having attenuated virulence, that confer protection fromvirulent mycobacteria, particularly M. tuberculosis. The instantinvention satisfies that need.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that deletion of the RD1region or a region controlling the production of a vitamin from thegenome of virulent mycobacteria in the M. tuberculosis complexattenuates the virulence of the mycobacteria without eliminating theability of the mycobacteria to colonize susceptible mammals. Theseattenuated mycobacteria are capable of protecting the mammals fromchallenge by a virulent M. tuberculosis complex mycobacteria. Theattenuated mycobacteria are thus useful in methods and compositions forvaccination of humans, cows and other mammals from virulent M.tuberculosis complex mycobacteria.

Accordingly, in some embodiments, the present invention is directed to anon-naturally occurring Mycobacterium tuberculosis. The M. tuberculosiscomprises a deletion of an RD1 region or a region controlling productionof a vitamin. The M. tuberculosis preferably exhibits attenuatedvirulence in a mammal when compared to the M. tuberculosis without thedeletion.

In certain aspects of these embodiments, the Mycobacterium tuberculosisis produced by deletion of an RD1 region or a region controllingproduction of a vitamin. In these aspects, the M. tuberculosis alsopreferably exhibits attenuated virulence in a mammal when compared tothe M. tuberculosis without the deletion.

In related embodiments, the present invention is also directed tomycobacteria in the M. tuberculosis complex that are geneticallyengineered to comprise a deletion of an RD1 region or a regioncontrolling production of a vitamin.

The present invention is also directed to mycobacteria in the M.tuberculosis complex that comprise a deletion of a region controllingproduction of a vitamin. These mycobacteria are preferably capable ofsustaining an infection in an immunocompetent mouse for at least 20weeks.

The inventors have also discovered that mycobacteria that areauxotrophic for lysine have attenuated virulence and can protect amammal against challenge by a virulent mycobacterium. Accordingly, theinvention is also directed to non-naturally occurring mycobacteria inthe M. tuberculosis complex, wherein the mycobacteria comprise adeletion of a region controlling production of lysine, and wherein themycobacteria are capable of sustaining an infection in animmunocompetent mouse for at least 20 weeks.

The inventors have additionally discovered that mycobacteria having twoattenuating deletions are highly attenuated, even in immunocompromisedmammals, and are surprisingly effective in protecting mammals againstchallenge by a virulent microorganism. Thus, the invention isadditionally directed to mycobacteria in the M. tuberculosis complexthat are genetically engineered to comprise two deletions. The twodeletions are any deletions where a virulent mycobacterium in the M.tuberculosis complex having either deletion exhibits attenuatedvirulence.

In further embodiments, the invention is directed to tuberculosisvaccines comprising any of the above-described M. tuberculosis ormycobacteria in the M. tuberculosis complex, in a pharmaceuticallyacceptable excipient. These vaccines are capable of protecting mammalsfrom challenge by virulent mycobacteria in the M. tuberculosis complex.

The invention is also directed to methods of protecting mammals fromvirulent M. tuberculosis or mycobacteria in the M. tuberculosis complex.The methods comprise treating the mammal with any of the above vaccines.

In other embodiments, the invention is directed to methods of preparingtuberculosis vaccines. The methods comprise deleting an RD1 region or aregion controlling production of a vitamin or lysine from an M.tuberculosis to produce any of the M. tuberculosis described above. Inthese embodiments, the vaccine is capable of protecting the mammal fromchallenge by a virulent M. tuberculosis.

In related embodiments, the invention is directed to other methods ofpreparing a tuberculosis vaccine. These methods comprise geneticallyengineering a mycobacterium to delete an RD1 region or a regioncontrolling production of a vitamin or lysine to produce any of themycobacteria described above. In these embodiments, the vaccine iscapable of protecting the mammal from challenge by a virulentmycobacteria of the M. tuberculosis complex.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows maps and autoradiographs pertaining to the construction ofΔRD1 mutants of M. tuberculosis. Panel a, M. tuberculosis H37Rvpublished sequence between 4346 kb and 4364 kb, showing predicted NcoIsites. Arrows on the top represent the genes in the RD1 region. The RD1region deleted from M. bovis BCG is represented by an open bar spanningfrom Rv3871 to Rv3879c. Upstream and downstream flanking sequences, UFSand DFS respectively, are indicated as closed bars underneath the gridline. Panel b, Southern hybridization of M. tuberculosis H37Rv ΔRD1created using two-step sequential homologous recombination. Panel c,Southern hybridization of M. tuberculosis H37Rv and Erdman ΔRD1 strainscreated using specialized transduction.

FIG. 2 shows graphs summarizing experiments establishing that M.tuberculosis H37Rv ΔRD1 is attenuated in SCID mice. Panel a, Sevenfemale SCID mice were infected intravenously with 2×10⁶ CFU M.tuberculosis H37Rv, M. tuberculosis H37Rv ΔRD1, and M. tuberculosisH37Rv ΔRD1::2F9 per mouse. The number of surviving mice was recordedpost infection. Panel b, Mice were infected with different doses of M.tuberculosis H37Rv, M. tuberculosis H37Rv ΔRD1, and M. bovis BCG. Foreach strain, infection doses of 2×10⁶ CFU, 2×10⁵ CFU, 2×10⁴ CFU, and2×10³ CFU per mouse, were administered via tail intravenous injection.

FIG. 3 is photographs, micrographs and autoradiographs showing that theM. tuberculosis H37Rv ΔRD1 mutant exhibits two distinct colonialmorphotypes. Panel a, M. tuberculosis H37Rv. Panel b, M. tuberculosisH37Rv ΔRD1. Panel c, M. tuberculosis H37Rv ΔRD1::2F9. Panel d, Southernanalysis of M. tuberculosis H37Rv ΔRD1 NcoI-digested genomic DNA,isolated from three smooth and three rough colonies and probed with DFS.Panels e-g, Colonial morphotypes at higher magnification. e, Smoothmorphotype at week 4. f, Rough morphotype at week 4. g, Rough morphotypeat week 6.

FIG. 4 is graphs showing the growth kinetics of M. tuberculosis H37RvΔRD1 in BALB/c mice. Mice were infected with 2×10⁶ CFU through tailinjection. Time to death was noted and at day 1, week 4, 8, 14, and 22post-infection, mice were sacrificed to determine the mycobacterialburden in the spleen, liver, and lung. The numbers represent the meansof CFUs in organs derived from three animals. The error bars representthe standard errors of the means. Panel a, Time to death assay in BALB/cmice. Panel b, Spleen. Panel c, Liver. Panel d, Lung.

FIG. 5 is micrographs from pathological studies of infected BALB/c mice.Panels a-c, Lungs from mice infected with 2×10⁶ CFU of M. tuberculosisH37Rv examined at 4, 8 and 14 weeks post-infection. The mild to moderatepneumonia at 4 and 8 weeks (a and b) progressed to severe consolidatinggranulomatous pneumonia at 14 weeks post infection (c). Panels d-f,Lungs from mice infected with 2×10⁶ CFU of M. tuberculosis H37Rv ΔRD1examined at 4, 8 and 22 weeks post-infection showing moderate pneumoniaat 8 weeks post-infection (e) and persistent bronchitis and multifocalpneumonitis at 22 weeks post-infection (f). Panels (g)-(i), Mild lunglesions from mice infected with 2×10⁶ CFU of BCG at 4, 8 and 22 weekspost-infection. Mild focal granulomas scattered widely in the lung ateach time point with predominately lymphocytic accumulations in foci at22 weeks post-infection.

FIG. 6 shows graphs summarizing experiments establishing thatpantothenate auxotrophy leads to attenuation of M. tuberculosis ΔpanCDmutant in SCID mice. Panel A, Survival of BALB/c SCID mice (n=12 pergroup) infected intravenously with 490 CFU of H37Rv (∘) or 210 CFU ofpanCD complementing strain (panCD in single copy integrated into thechromosome)(●) or 3.4×10⁵ CFU of ΔpanCD mutant (▴) or 3.3×10⁵ CFU ofBCG-P (□). Panel B, Bacterial numbers in the spleen (●) and lungs (▴) ofSCID mice infected intravenously with 490 CFU of H37Rv or numbers inspleen (∘) and lungs (Δ) of mice infected with 3.4×10⁵ CFU of ΔpanCDmutant. The results represent means± standard errors of four to fivemice per group.

FIG. 7 shows graphs summarizing experiments demonstrating theattenuation, limited growth and persistence of ΔpanCD mutant inimmunocompetent mice. Panel A, Survival of BALB/c mice (n=12 per group)infected with 4.4×10⁶ CFU of wild-type M. tuberculosis H37Rv (∘),3.2×10⁶ CFU panCD complementing strain (panCD in single copy integratedinto the chromosome)(●) or 2.4×10⁶ CFU panCD mutant (▴). Panels B and C,Bacterial loads in spleen and lungs of BALB/c mice infectedintravenously with 4.4×10⁶ CFU wild-type H37Rv (∘) or 3.2×10⁶ CFU panCDcomplementing strain (●) or 2.4×10⁶ CFU ΔpanCD mutant (▴). CFUs wereassayed at various time points on 7H11 agar with or without pantothenatesupplementation where required. The results represent means± standarderrors of four to five mice per group.

FIG. 8 shows graphs summarizing experiments demonstrating theattenuation, limited replication and persistence of ΔnadBC mutant inimmunocompetent mice. Panels A and B, Bacterial loads in lungs andspleen of C57BL/6 mice infected with wild type M. tuberculosis H37Rv (●)or ΔnadBC mutant (∘). Mice were infected intravenously with 10⁶ CFU ofeach strain. CFUs were assayed at various time points on 7H11 agar withor without nicotinamide supplementation where required. The resultsrepresent means± standard errors of four to five mice per group. PanelC, Survival of C57BL/6 mice (n=12 per group) infected with 10⁶ CFU ofwild-type bacteria (●) or 10⁶ CFU of ΔnadBC mutant (∘).

FIG. 9 shows an illustration, map and autoradiograph relating to thepathway for the biosynthesis of pantothenic acid and coenzyme A and itsdisruption in M. tuberculosis. Panel a. The enzymes involved in thebiosynthesis of pantothenic acid and having annotation in the genomicsequence of M. tuberculosis H37Rv are shown in bold numbers: 1) panB,ketopantoate hydroxymethyl transferase; 2) panD,aspartate-1-decarboxylase; 3) panC, pantothenate synthetase; 4) panK,pantothenate kinase; 5) acpS, ACP synthase. Panel b. Map of the panCDgenomic region in the wild type M. tuberculosis H37Rv and the ΔpanCDmutant. Restriction sites and probe location are indicated. Panel c.Southern blot of BssHII-digested genomic DNA from wild-type H37Rv (lane1), two independent clones of ΔpanCD mutant from H37Rv (lanes 2 & 3) andprobed with a 716 bp downstream region flanking the panCD operon.Molecular size marker (in kb) is shown on the left.

FIG. 10 shows graphs summarizing experiments demonstrating thatpantothenate auxotrophy leads to attenuation of ΔpanCD mutant in mice.a. Survival of BALB/c SCID mice (n=12 per group) infected intravenouslywith H37Rv (∘) or panCD-complemented strain (●) or ΔpanCD mutant (▴) orM. bovis BCG-P (□). b. Bacterial numbers in the spleen (∘), liver (□)and lung (Δ) of SCID mice infected intravenously with H37Rv or thebacterial numbers in the spleen (●), liver (▪) and lung (▴) of miceinfected with ΔpanCD mutant. c. Survival of immunocompetent BALB/c mice(n=16 per group) infected with H37Rv (∘) or panCD-complemented strain(●) or ΔpanCD mutant (▴). d, e, f. Bacterial numbers in lung (d), spleen(e) and liver (f) of immunocompetent BALB/c mice infected intravenouslywith either H37Rv (∘), panCD-complemented strain (●) or ΔpanCD mutant(▴). Data are means± standard errors of four to five mice per group.

FIG. 11 shows micrographs (Panels a-d) and graphs (Panels e and f)summarizing experiments demonstrating that the ΔpanCD mutant producesless tissue pathology in lungs of infected BALB/c mice and protects miceagainst challenge with virulent M. tuberculosis. Panel a. Severeconsolidating granulomatous pneumonia (★) obliterating the normal lungparenchyma at 3 weeks post-infection with H37Rv. Panel b. Severeconsolidating granulomatous pneumonia (★) obliterating the normal lungparenchyma at 3 weeks post-infection with the panCD-complemented strain,similar to the wild type strain. Panel c. Mild lung infection caused bythe ΔpanCD mutant at 3 weeks post-infection. Localized multifocalgranulomas (arrows) scattered widely in the lung. Most of the lung isnormal alveolar spaces and airways. Panel d. Lung of mouse infected withΔpanCD mutant examined histologically at 23 weeks post-infection.Occasional focal, mild perivascular and interstitial infiltrationscomposed of predominately lymphocytes (arrows). Most of the lung isnormal alveolar spaces and airways. e, f. The attenuated ΔpanCD mutantprotects mice against aerogenic challenge with virulent M. tuberculosisErdman. Subcutaneously immunized mice were challenged after 90 daysthrough the aerosol route. The CFU numbers reflect the bacterial burdenat 28 days post aerosol challenge in the lung (e) and spleen (f). Naivemice—black fill; mice infected with 1 dose panCD—light shade; miceinfected with 2 doses panCD—dark shade; mice infected withBCG-P—unshaded.

FIG. 12 shows autoradiographs of Southern analysis of the NcoI-digestedgenomic DNA isolated from the wild type and the ΔRD1 mutants generatedusing specialized transduction in M. tuberculosis and M. bovis. Lanes:1—M. tuberculosis H37Rv; 2—M. tuberculosis H37Rv ΔRD1; 3—M. tuberculosisErdman; 4—M. tuberculosis Erdman ΔRD1; 5—M. tuberculosis CDC1551; 6—M.tuberculosis CDC1551 ΔRD1; 7—M. bovis Ravenel; and 8—M. bovis RavenelΔRD1. The probes used in the Southern analysis was either DFS (left) orIS6110-specific sequence (right).

FIG. 13 shows graphs summarizing data confirming that deletion of RD1 inM. tuberculosis and M. bovis confers an attenuation of virulence for M.tuberculosis and M. bovis, as indicated by these Time to death curves ofmice infected intravenously with 2×10⁶ CFU mycobacteria. Panel A, SCIDmice infected with M. tuberculosis H37Rv (▪), M. tuberculosis H37Rv ΔRD1(□), M. tuberculosis Erdman (●), M. tuberculosis Erdman ΔRD1 (∘), M.tuberculosis CDC1551 (▴), M. tuberculosis CDC1551 ΔRD1 (Δ), M. bovisRavenel (▾), M. bovis Ravenel ΔRD1 (∇); Panel B, SCID mice infectedintravenously with M. tuberculosis H37Rv (●), M. tuberculosis ΔRD1 (▪),M. tuberculosis ΔRD1::2F9 (▴), M. bovis Ravenel (∘), M. bovis RavenelΔRD1 (□), and M. bovis BCG (Δ); Panel C, BALB/c mice were infected withM. tuberculosis H37Rv (∘), M. tuberculosis ΔRD1 (Δ), and M. bovis BCG(□).

FIG. 14 are graphs summarizing experiments demonstrating the clearanceof the lysine auxotroph in SCID mice. The viable bacterial counts areshown for the spleens, livers, and lungs of SCID mice injectedintravenously with the lysine auxotroph strain and the prototrophiccontrol strain. Three mice were assayed at each time point. The errorbars indicate the standard deviations of the mean values. Note that thecounts at time zero are the counts obtained at 24 hours post-injection,as described in Example 5. Panels A, B and C show the log of the viablebacteria in each organ after injection with 1×10⁷ CFU of the Lys⁻ M.tuberculosis mutant mc²3026 (□), or 1×10⁷ CFU of the complemented Lys⁺M. tuberculosis strain mc²3026/pYUB651 (▪).

FIG. 15 is graphs summarizing experimental results of experiments thatestablish the vaccine efficacy of the M. tuberculosis lysine auxotrophmc²3026. C57B1/6 mice were injected intravenously with 1×10⁶ CFU of theM. tuberculosis lysine auxotroph mc²3026, followed by one or twoadditional injections at 4 week intervals. Five mice were sacrificedweekly after each immunization and the viable bacteria counts of theauxotroph determined in the lungs and spleens. Control mice were given asimilar amount of BCG-Pasteur or only PBST. Shown in Panel A is theclearance of the auxotroph from the lungs of the mice after eachimmunization period; one injection (▪), two injections (♦), and threeinjections (●). Three months after the initial immunization thevaccinated and control mice were challenged with virulent M.tuberculosis Erdman by the aerosol route. Five challenge mice weresacrificed following the challenge period and the lung homogenatesplated to check the viable counts of the challenge inoculum. Groups ofvaccinated and control mice were sacrificed at 14, 28, and 42 days laterand the lung and spleen homogenates plated to determine viable colonyforming units. Shown in Panel B are the viable challenge bacteria perlung of mice given one dose of the M. tuberculosis lysine auxotroph, andin panel C, the viable challenge bacteria per lung of mice given twodoses of the auxotroph. Key: Viable challenge bacteria per lung of micegiven the M. tuberculosis lysine auxotroph mc²3026 (▪), BCG-Pasteur (⋄),or PBST (∘). P values are indicated in the figure. Note that the resultsshown here are for the lungs. Similar results (not shown) were obtainedfrom the spleens in all the experiments.

FIG. 16 shows a graph summarizing experiments establishing the survivalcurves of mice immunized three times with the M. tuberculosis lysineauxotroph mc²3026. C57B1/6 mice were injected intravenously with 1×10⁶CFU of the M. tuberculosis lysine auxotroph mc²3026, followed by twomore injections at 4 week intervals, and challenged as described inExample 5. The percent survival is shown for mice immunized thrice withthe M. tuberculosis lysine auxotroph mc²3026 (▪, 5 mice total), oncewith BCG-Pasteur (♦, 5 mice), and for the PBST controls (●, 10 mice).

FIG. 17 shows graphs summarizing experimental results establishing thatthe virulence of strain mc²6030 is highly attenuated in SCID mice andBALB/c mice.

FIG. 18 shows graphs summarizing experimental results measuring growthof various strains of M. tuberculosis in spleen (Panel A) and lungs(Panel B) of C57BL/6 mice.

FIG. 19 is a graph summarizing experimental results establishing thatimmunization with mc²6020 and mc²6030 protects mice against TB aseffectively as BCG. This graph shows the survival of C57BI/6 micechallenged with virulent M. tuberculosis Erdman through the aerosolroute three months after a single dose subcutaneous immunization witheither BCG, mc²6020 (ΔlysAΔpanCD) or mc²6030 (ΔRD1ΔpanCD) and comparedto non-immunized naive mice. There were 12 to 15 mice in each survivalgroup.

FIG. 20 shows graphs summarizing experimental results establishing thatM. tuberculosis double deletion mutants are highly attenuated in SCIDmice. A dose of 10⁵ mc²6020 or mc²6030 were intravenously inoculatedinto SCID mice (10 per group) and time to death assessments wereperformed. While the same dose of M. tuberculosis and BCG killed mice in40 or 90 days, respectively, the mice infected with mc²6020 or mc²6030survived over 400 or 250 days, respectively.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based in part on the discovery that virulentmycobacteria in the M. tuberculosis complex that have deletions in theRD1 region, or in a region that controls production of a vitamin, areattenuated in virulence but are capable of sustaining viability andgrowth in a mammalian host, and are also capable of protecting against achallenge by a virulent M. tuberculosis complex mycobacterium.

Thus, in some embodiments, the invention is directed to non-naturallyoccurring Mycobacterium tuberculosis that comprise a deletion of an RD1region or a region controlling production of a vitamin. These M.tuberculosis preferably exhibit attenuated virulence in a mammal whencompared to the M. tuberculosis without the deletion.

A host organism can be inoculated with the mycobacteria of the presentinvention by any of a number of ways known in the art. These includeoral ingestion, gastric intubation, or broncho-nasal-ocular spraying.Other methods of administration include intravenous, intramuscular,intramammary, or, preferably, subcutaneous or intradermal injection. Theimmunization dosages required can be determined without undueexperimentation. One or two dosages of avirulent mycobacteria at 1-2×10⁶colony forming units (CFU) have previously been used, but other dosagesare contemplated within the scope of the invention. Multiple dosages canbe used as needed to provide the desired level of protection fromchallenge.

It is well known in the art that in order to elicit an immune responsewith a live vaccine such as an avirulent mycobacteria, it is preferredthat the vaccine organism can sustain an infection in the immunizedhost, to provide a sustained exposure of the host's immune system to theorganism. Therefore, in various preferred embodiments, the M.tuberculosis of the invention are capable of sustaining an infection inthe host. The ability to sustain infection can be measured without undueexperimentation by any of a number of ways described in the art. Withthe mycobacteria of the present invention, a preferred way of measuringsustained infection is by determining whether viable mycobacteria of theinoculated strain will remain resident in an immunocompetent mouse(e.g., BALB/c or C57BL/6 strain) for more than four weeks. Morepreferably, the inoculated mycobacteria will remain resident in themouse for at least ten weeks. In the most preferred embodiments, viablemycobacteria of the inoculated strain will remain resident in the mousefor at least 20 weeks.

Preferably, the attenuated mycobacteria of the invention are capable ofprotecting a mammal from challenge by a virulent M. tuberculosis complexmycobacteria. This ability can be determined by any of a number of waysprovided in the literature. A preferred method is aerogenically treatingan immunocompetent mouse with the virulent mycobacteria, as described inExamples 1 and 2. Aerogenic challenge is preferred because that mostclosely mimics natural infection. The skilled artisan would understandthat the ability of an avirulent mycobacterium to protect a mouse fromchallenge from a virulent mycobacterium is indicative of the ability ofthe avirulent mycobacterium to protect a human, including a human child,from tuberculosis infection. A more stringent test of an avirulentmycobacterium to prevent infection by a virulent challenge is to use animmunocompromised mammal such as a SCID mouse.

The deletion of the RD1 region or the region controlling production of avitamin is contemplated in these embodiments with any M. tuberculosisstrain. Preferably, the strain is a virulent strain, since those strainswould be most likely to sustain an infection after the deletion is made.Preferred M. tuberculosis strains are the H37Rv and CDC1551 strain,because the genetics of those strains are very well known.

In some aspects of these embodiments, the deletion is of the RD1 region(see Example 1). Strains with these deletions can be determined by anymeans in the art, preferably by molecular genetic means, for example byhybridization methods (e.g., Southern blot using a probe from the RD1region) or by amplification methods (e.g., PCR using primers to amplifya portion of the RD1 region). An example of an M. tuberculosis RD1region (from H37Rv) is provided herein as SEQ ID NO:1. The skilledartisan could identify analogous RD1 regions from other M. tuberculosiscomplex mycobacteria without undue experimentation. Those RD1 regionswould be expected to have strong homology to SEQ ID NO:1, at least 80%homologous to SEQ ID NO:1. However, it is to be understood that virulentM. tuberculosis can be rendered avirulent by deletions in a portion ofthe RD1 region. Therefore, non-naturally occurring M. tuberculosis thathave a partial deletion in the RD1 region are envisioned as within thescope of the invention, provided the deletion can cause a virulent M.tuberculosis to become avirulent. It is expected that such M.tuberculosis with partial RD1 deletions can still sustain an infectionin a mammal and protect against challenge by a virulent M. tuberculosis.

In embodiments where the deletion is in a region controlling productionof a vitamin, the deletion can be in any genetic element leading to lossof production of the vitamin, including structural genes for enzymesinvolved in the biosynthesis of the vitamin, and genetic controlelements such as promoters, enhancers, etc.

Deletion of a region controlling production of any essential vitamin ortheir precursors is contemplated as within the scope of the invention.As used herein, an essential vitamin is defined by its normal usage,that is, a small molecular weight compound that is required as acofactor for the efficient function of an essential enzyme or enzymes.Nonlimiting examples include vitamin A, thiamin (B1), riboflavin (B2),nicotinic acid (niacin)/nicotinamide/nicotinamide adenine dinucleotide(NAD)/nicotinamide adenine dinucleotide phosphate (NADP/coenzyme II),pantothenate (pantothenic acid/B5), pyridoxine (B6), folic acid, B12,biotin, C, D, E and K. Preferred vitamin targets for deletion includenicotinamide and pantothenate (see Example 2). Methods for determiningwhether a mycobacterium has deletions leading to the loss of productionof any of these vitamins are within the scope of the art.

Deletions leading to the loss of any of these vitamins would be expectedto lead to attenuated virulence of an otherwise virulent mycobacteriumin the M. tuberculosis complex. Any of those strains would also beexpected to sustain an infection in a mammal.

Preferred vitamin targets are pantothenate and nicotinamide adeninedinucleotide (NAD) (see Example 2). A preferred pantothenate deletion isof structural genes in the pantothenate biosynthetic operon, mostpreferably the panC and panD genes, the combined mutation being ΔpanCD.An example of a deletion of those genes is the deletion of the sequencefrom M. tuberculosis H37Rv provided herein as SEQ ID NO:2. Similarly, apreferred NAD deletion is in the structural genes of the NADbiosynthetic operon, most preferably the nad B and C genes, the combinedmutation being ΔnadBC. An example of a deletion in those genes is thedeletion of the sequence from M. tuberculosis H37Rv provided herein asSEQ ID NO:3.

In similar embodiments, the invention is directed to any of theabove-described M. tuberculosis that are produced by deleting an RD1region or a region controlling production of a vitamin. The deletion canbe made by serial in vitro passage of a virulent M. tuberculosis (as thewell-known M. bovis BCG was made) and selection for the desireddeletion. More preferably, however, the deletion is made by geneticengineering, since such genetic methods allow precise control of thedeletion being made.

Various methods of making deletions in mycobacteria are known in theart. Nonlimiting examples include specialized transduction (see, e.g.,U.S. Pat. No. 6,271,034, Example 1 and Example 2), and sequentialtwo-step recombination (see Example 1). The latter method can usefullyemploy a sacB selective marker (Example 1).

Since, in preferred embodiments of the invention, the mycobacteriaexhibit attenuated virulence and can sustain an infection in a mammal,these mycobacteria can usefully further employ a foreign DNA stablyintegrated into the genome of the mycobacteria, such that themycobacteria can express a gene product coded by the foreign DNA. See,e.g., U.S. Pat. No. 5,504,005.

Thus, it is apparent that the present invention has wide applicabilityto the development of effective recombinant vaccines against bacterial,fungal, parasite or viral disease agents in which local immunity isimportant and might be a first line of defense. Non-limiting examplesare recombinant vaccines for the control of bubonic plague caused byYersinia pestis, of gonorrhea caused by Neisseria gonorrhoea, ofsyphilis caused by Treponema pallidum, and of venereal diseases or eyeinfections caused by Chlamydia trachomatis. Species of Streptococcusfrom both group A and group B, such as those species that cause sorethroat or heart disease, Neisseria meningitidis, Mycoplasma pneumoniae,Haemophilus influenzae, Bordetella pertussis, Mycobacterium leprae,Streptococcus pneumoniae, Brucella abortus, Vibrio cholerae, Shigellaspp., Legionella pneumophila, Borrelia burgdorferi, Rickettsia spp.,Pseudomonas aeruginosa, and pathogenic E. coli such as ETEC, EPEC, UTEC,EHEC, and EIEC strains are additional examples of microbes within thescope of this invention from which foreign genes could be obtained forinsertion into mycobacteria of the invention. Recombinant anti-viralvaccines, such as those produced against influenza viruses, are alsoencompassed by this invention. Recombinant anti-viral vaccines can alsobe produced against viruses, including RNA viruses such asPicornaviridae, Caliciviridae, Togaviridae, Flaviviridae, Coronaviridae,Rhabdoviridae, Filoviridae, Paramyxoviridae, Orthomyxoviridae,Bunyaviridae, Arenaviridae, Reoviridae or Retroviridae; or DNA virusessuch as Hepadnaviridae, Paroviridae, Papovaviridae, Adenoviridae,Herpesviridae or Poxyiridae.

Recombinant vaccines to protect against infection by pathogenic fungi,protozoa or parasites are also contemplated by this invention.

The avirulent microbes of the present invention are also contemplatedfor use to deliver and produce foreign genes that encodepharmacologically active products that might stimulate or suppressvarious physiological functions (i.e., growth rate, blood pressure,etc.). In such microbes, the recombinant gene encodes saidpharmacologically active products.

By immunogenic agent is meant an agent used to stimulate the immunesystem of an individual, so that one or more functions of the immunesystem are increased and directed towards the immunogenic agent.Immunogenic agents include vaccines.

An antigen or immunogen is intended to mean a molecule containing one ormore epitopes that can stimulate a host immune system to make asecretory, humoral and/or cellular immune response specific to thatantigen.

In preferred embodiments, the foreign DNA encodes an antigen, an enzyme,a lymphokine, an immunopotentiator, or a reporter molecule. Preferredexamples include antigens from Mycobacterium leprae, Mycobacteriumtuberculosis, malaria sporozoites, malaria merozoites, diphtheriatoxoid, tetanus toxoids, Leishmania spp., Salmonella spp., Mycobacteriumafricanum, Mycobacterium intracellulare, Mycobacterium avium, Treponemaspp., Pertussis, Herpes virus, Measles virus, Mumps virus, Shigellaspp., Neisseria spp., Borrelia spp., rabies, polio virus, humanimmunodeficiency virus, snake venom, insect venom, and Vibrio cholera;steroid enzymes; interleukins 1 through 7; tumor necrosis factor α andβ; interferon α, β, and γ; and reporter molecules luciferase,β-galactosidase, β-glucuronidase and catechol dehydrogenase.

The scope of the present invention includes novel mycobacteria in the M.tuberculosis complex that are genetically engineered to comprise adeletion of an RD1 region or a region controlling production of avitamin. The scope of the deletions and the characteristics of thesemycobacteria are as with the M. tuberculosis mycobacteria describedabove. These mycobacteria include any in the M. tuberculosis complex,including M. africanum, M. bovis including the BCG strain and thesubspecies caprae, M. canettii, M. microti, M. tuberculosis and anyother mycobacteria within the M. tuberculosis complex, now known orlater discovered. Preferred species are M. bovis, including the BCGstrain, and M. tuberculosis, since those species are the most importantas causes of mammalian diseases, such as tuberculosis in humans and M.bovis infection in cows.

Also included as within the scope of the invention is any non-naturallyoccurring mycobacterium in the M. tuberculosis complex having a deletionof a region controlling production of a vitamin. These mycobacteriapreferably are capable of sustaining an infection in a mammal. The scopeof the deletions and the characteristics of these mycobacteria are aswith the M. tuberculosis and other mycobacteria described above.

The inventors have also discovered that mycobacteria in the M.tuberculosis complex that are auxotrophic for lysine have attenuatedvirulence and protect a mammal from challenge by a virulentmycobacterium. See Example 5. Thus, in some embodiments, the inventionis directed to non-naturally occurring mycobacteria in the M.tuberculosis complex that comprise a deletion of a region controllingproduction of lysine. These mycobacteria are capable of sustaining aninfection in an immunocompetent mouse for at least 20 weeks. As withpreviously described embodiments, these mycobacteria can be any speciesin the M. tuberculosis complex. However, due to their importance asdisease organisms, it is preferred mycobacteria are M. tuberculosis andM. bovis, e.g., M. bovis BCG.

These mycobacteria would also be expected to exhibit attenuatedvirulence in a mammal when compared to the mycobacteria without thedeletion. Additionally, they would be expected to provide protection toa mammal from challenge by a virulent mycobacterium in the M.tuberculosis complex. A preferred deletion is a ΔlysA deletion, forexample as provided herein as SEQ ID NO:4.

When constructing a live vaccine that is an attenuated pathogen due to adeletion, it is often desirable to include a second deletion, to betterassure the safety of the vaccine. Second deletions in any of theabove-described mycobacteria are thus contemplated as within the scopeof the invention. The second deletion preferably can also attenuatevirulence of an otherwise virulent mycobacterium in the M. tuberculosiscomplex. This second deletion can be the RD1 region if the firstdeletion is not. The second deletion can also be a deletion that wouldcause a prototrophic mycobacterium to be auxotrophic, or any otherdeletion that could improve the safety or efficacy of the mycobacteriumin protecting against infection. Nonlimiting examples include deletionsin a gene or genes controlling production of an amino acid or anucleotide, or a vitamin not eliminated by the first mutation.

The inventors have also discovered that two attenuating deletions in amycobacterium in the M. tuberculosis complex provides a high level ofprotection to a mammal from challenge by a virulent mycobacterium. SeeExample 6.

Thus, in some embodiments, the invention is directed to mycobacteria inthe M. tuberculosis complex which are genetically engineered to comprisetwo deletions. Preferably, each of the two deletions are capable ofindividually attenuating virulence when engineered into a virulentmycobacterium in the M. tuberculosis complex.

Preferred embodiments of these mycobacteria are as with the othermycobacteria of the invention, e.g., the mycobacterium is preferably aMycobacterium tuberculosis; the mycobacterium is preferably capable ofsustaining an infection in an immunocompetent mouse for at least 20weeks; and the mycobacterium is capable of protecting the mammal fromchallenge by a virulent mycobacterium.

As with the other mycobacteria previously described, the two attenuatingdeletions can be any deletions that are individually capable ofattenuating virulence of an otherwise virulent strain. Preferreddeletions are deletions of an RD1 region (e.g., a deletion of SEQ IDNO:1), deletions of a region controlling production of a vitamin, ordeletions of a region controlling the production of an amino acid, aspreviously discussed. A preferred deletion of a region controllingproduction of a vitamin is the ΔpanCD deletion, e.g., as disclosed inExamples 2 and 3, discussing attenuated strains having a deletion of SEQID NO:2. Preferred deletions of regions controlling production of aminoacids are those regions controlling production of proline, tryptophan,leucine or lysine. See, also, Examples 5 and 6, describing strainshaving a ΔlysA deletion (SEQ ID NO:4), or two mutations including onewith a ΔlysA deletion.

In additional embodiments, the invention is directed to tuberculosisvaccines made using any of the above described mycobacteria, in apharmaceutically acceptable excipient. These vaccines are capable ofprotecting the mammal from challenge by a virulent M. tuberculosiscomplex mycobacteria. In some preferred embodiments, the mycobacteriumis a Mycobacterium bovis and the mammal is a cow; in other preferredembodiments, the mycobacterium is M. tuberculosis and the mammal is ahuman, e.g., a human child.

By vaccine is meant an agent used to stimulate the immune system of anindividual so that protection is provided against an antigen notrecognized as a self-antigen by the immune system. Immunization refersto the process of inducing a continuing high level of antibody and/orcellular immune response in which T-lymphocytes can either kill thepathogen and/or activate other cells (e.g., phagocytes) to do so in anindividual, which is directed against a pathogen or antigen to which theorganism has been previously exposed. The phrase “immune system” refersherein to the anatomical features and mechanisms by which a mammalproduces antibodies against an antigenic material which invades thecells of the individual or the extra-cellular fluid of the individualand is also intended to include cellular immune responses. In the caseof antibody production, the antibody so produced can belong to any ofthe immunological classes, such as immunoglobulins, A, D, E, G or M.Immune responses to antigens are well studied and widely reported. Asurvey of immunology is provided in Elgert (1996) and Stites et al.(1991).

The pharmaceutical carrier or excipient in which the vaccine issuspended or dissolved may be any solvent or solid or encapsulatingmaterial. The carrier is non-toxic to the inoculated individual andcompatible with the microorganism or antigenic gene product. Suitablepharmaceutical carriers are known in the art and, for example, includeliquid carriers, such as normal saline and other non-toxic salts at ornear physiological concentrations, and solid carriers, such as talc orsucrose. Gelatin capsules can serve as carriers for lyophilizedvaccines. Adjuvants may be added to enhance the antigenicity if desired.When used for administering via the bronchial tubes, the vaccine ispreferably presented in the form of an aerosol. Suitable pharmaceuticalcarriers and adjuvants and the preparation of dosage forms are describedin, for example, Gennaro (1985).

Similarly, the invention is directed to methods of protecting a mammalfrom a virulent mycobacterium in the M. tuberculosis complex. Themethods comprise treating the mammal with any of the above-describedvaccines.

The vaccines can be administered by oral ingestion, gastric intubation,or broncho-nasal-ocular spraying, intravenous, intramuscular,intramammary, or, preferably, by subcutaneous or intradermal injection.The immunization dosages required can be determined without undueexperimentation. One or two dosages of avirulent mycobacteria at 1-2×10⁶colony forming units (CFU) have previously been used, but other dosagesare contemplated within the scope of the invention. Multiple dosages canbe used as needed to provide the desired level of protection fromchallenge (see, e.g., Example 5).

The present invention is also directed to methods of preparing atuberculosis vaccine. The methods comprise deleting an RD1 region or aregion controlling production of a vitamin from a mycobacterium in theM. tuberculosis complex to produce any of the mycobacteria previouslydescribed.

Preferred embodiments of the invention are described in the followingexamples. Other embodiments within the scope of the claims herein willbe apparent to one skilled in the art from consideration of thespecification or practice of the invention as disclosed herein. It isintended that the specification, together with the examples, beconsidered exemplary only, with the scope and spirit of the inventionbeing indicated by the claims which follow the examples.

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Maniatis, Fritsch & Sambrook,“Molecular Cloning: A Laboratory Manual” (1989); “Current Protocols inMolecular Biology” Volumes I-IV (Ausubel, R. M., ed. (1997); and “CellBiology: A Laboratory Handbook” Volumes I-III (J. E. Celis, ed. (1994).

EXAMPLE 1 Mycobacterium Tuberculosis Having an RD1 Deletion hasAttenuated Virulence and Protects Against Tuberculosis

This example describes experimental methods and results that establishthat deleting the RD1 region from a virulent M. tuberculosis attenuatesthe virulence of the M. tuberculosis in both immunocompetent andimmunocompromised mice, and protects against subsequent challenge by avirulent M. tuberculosis.

Materials and Methods

Media and Cultures. The mycobacterial strains M. tuberculosis H37Rv, M.tuberculosis Erdman and M. bovis BCG Pasteur were obtained from theTrudeau Culture Collection (Saranac Lake, N.Y.). They were cultured inMiddlebrook 7H9 broth and 7H10 agar supplemented with 10% OADC, 0.5%glycerol, and 0.05% Tween 80. Cyclohexamide, which does not affectmycobacterial growth, was added to the 7H10 agar medium at 0.1% to avoidfungal contamination. To examine the colony morphology of mycobacteria,Tween 80 was not added to 7H10 agar medium. The acriflavin resistantstrain (Hepper and Collins, 1984) of M. tuberculosis Erdman grew in thepresence of 20 μg of acriflavin per ml of medium.

DNA manipulation and construction of M tuberculosis ΔRD1. The followingfour primers were used to amplify upstream and downstream flankingsequences (UFS and DFS, respectively) for the construction of the RD1deletion mutants. UFS was amplified using TH201: GGGGGCGCACCTCAAACC (SEQID NO:5) and TH202: ATGTGCCAATCGTCGACCAGAA (SEQ ID NO:6). DFS wasamplified using TH203: CACCCAGCCGCCCGGAT (SEQ ID NO:7), and TH204:TTCCTGATGCCGCCGTCTGA (SEQ ID NO:8). Recognition sequences for differentrestriction enzymes were included at the ends of each primer to enableeasier manipulation.

The unmarked deletion mutant of M. tuberculosis H37Rv, mc²4004, wasgenerated by transformation (Snapper et al., 1988) using a sacBcounterselection (Pelocic et al., 1996; Pavelka and Jacobs, 1999).Specifically, the plasmid pJH508 was created by first cloning UFS intoKpnI and XbaI sites, then cloning DFS into EcoRI and HindIII sites ofpJH12, a pMV261-derived E coli-Mycobacteria shuttle plasmid, to createpJH506 in which UFS and DFS flanked a green fluorescent protein gene(GFPuv, Clonetech) whose expression was driven by the M. leprae promoter18Kd. The UFS-gfp-DFS cassette was sub-cloned into the EcoRV site ofplasmid pYUB657 to create pJH508. The first homologous recombinationinvolved the identification of hygromycin resistant colonies, resultingfrom the transformation of M. tuberculosis with pJH508. Southernanalysis of the NcoI digested DNA isolated from hygromycin resistantcolonies probed with UFS or DFS, confirmed the presence of a single copyof pJH508 inserted into the M. tuberculosis genome. The transformantidentified was then grown in 7H9 broth to saturation, to allow thesecond homologous recombination to occur, resulting in recombinants thatcould be selected by plating the culture on 7H10 plates, supplementedwith 3% sucrose. Both Southern analysis and PCR of the DNA isolated fromsucrose resistant colonies confirmed the RD1 deletion.

Specialized transduction (Bardarov and Jacobs, 1999), amycobacteriophage-based method for the delivery of homologous DNAconstructs using conditionally replicating shuttle phasmids (Jacobs etal, 1987; Bardarov and Jacobs, 1999; Carriere et al., 1997), has beenused successfully for M. tuberculosis (Glickman et al., 2000; Glickmanet al., 2001; Raman et al., 2001). Specifically, a transducing phagephAEKO1 was constructed by inserting UFS and DFS into pJSC347, flankinga hygromycin cassette, to create pJH313. pJH313 was digested with PacIand ligated to phAE159, a temperature sensitive mycobacteriophagederived from TM4. The transduction was performed by growing M.tuberculosis to an O.D.₆₀₀ of 0.8, washing twice with MP buffer,re-suspending into an equal volume of MP buffer and mixing with thetransducing phage phAEKO1 at an MOI of 10. The mixtures were incubatedat 37° C. overnight, then plated on 7H10 plates supplemented withhygromycin at 50 μg/ml. Hygromycin resistant colonies were analyzed byPCR and Southern hybridization, as described above, to confirm thedeletion of RD1.

Complemetation analyses was performed using the integration proficientcosmids (Pascopella et al., 1994; Lee et al., 1991) pYUB412 made by S.Bardarov, a library made by F. Bange, and cosmid identified andgenerously provided by S. T. Cole.

Results

Genetic engineering of M. tuberculosis mutants with RD1 deletions. TheRD1 (region of difference) region has been defined as the specific 9454bp of DNA that is present in virulent M. tuberculosis and M. bovis, butabsent in BCG (Mahairas et al., 1996). The annotation of RD1 predictsthat the deletion would disrupt 9 genes encoding ORF's (Id.; Cole etal., 1998). Five of the 9 ORF's have no known functions (Rv3871, Rv3876,Rv3877, Rv3878 and Rv3879c), two genes encode members of the PE/PPEfamily (Rv3872/Rv3873), and two genes encode the secreted proteins Cfp10(Berhet et al., 1998) and Esat6 (Andersen et al., 1991) (Rv3875) (FIG.1). To test if the RD1 region is essential for virulence in M.tuberculosis, it was necessary to 1) delete the RD1 region from virulentM. tuberculosis strains, 2) demonstrate loss of virulence and 3) restorevirulence by complementation with the RD1 DNA. The RD1 deletion (ΔRD1)was successfully introduced into M. tuberculosis by two differenttechniques, utilizing both a plasmid that allows two-step sequentialrecombination to make an unmarked deletion, and specialized transduction(FIG. 1 a-c). For both methods, the same 1200 bp on each side of the RD1deletion were cloned into the appropriate plasmid or phage vector andthen introduced into M. tuberculosis H37Rv by transformation or phageinfection. An unmarked RD1 deletion mutant of M. tuberculosis H37Rv,mc²4004, was constructed, purified, and has the advantage thatadditional mutations can be readily added to it. In addition, the RD1deletion was successfully engineered in the H37Rv and Erdman strains ofM. tuberculosis using a specialized transducing phage. Since TM4 phageshave been shown to infect over 500 clinical M. tuberculosis isolates(Jacobs et al., 1987), it should be possible to introduce the RD1deletion into any M. tuberculosis isolate of interest.

M. tuberculosis H37Rv ΔRD1 is attenuated for virulence. To test if theRD1 deletion causes an attenuating phenotype in M. tuberculosis, the M.tuberculosis H37Rv ΔRD1 (mc²4004) was introduced into immunocompromisedmice possessing the SCID (severe combined immunodeficiency) mutation.Groups of ten mice were injected intravenously with either 2×10⁶ M.tuberculosis H37Rv or M. tuberculosis H37Rv ΔRD1 and three mice pergroup were sacrificed 24 hours later to verify the inoculation doses.All of the SCID mice infected with the parental M. tuberculosis H37Rvstrain died within 14 to 17 days post infection (FIG. 2 a). In contrast,the SCID mice infected with the same dose of M. tuberculosis H37Rv ΔRD1were all alive at 35 days post-infection demonstrating a markedattenuation of the strain. To prove that the attenuation was due to theRD1 deletion, mc²4004 was transformed with an integrating plasmidcontaining the RD1 region from M. tuberculosis H37Rv. SCID mice injectedintravenously with 2×10⁶ of the transformed strain died 13 to 16 dayspost-infection (FIG. 2 a), thereby, establishing that the genes in theRD1 deletion complemented the attenuating phenotype.

To further characterize the attenuating phenotype of the RD1 deletion inmc²4004, we compared the virulence of M. tuberculosis H37Rv andBCG-Pasteur to M. tuberculosis H37Rv ΔRD1 with time-to-death experimentsin SCID mice following injections with 10-fold varying inocula. Groupsof 10 mice were injected intravenously, each mouse receiving from 2×10³to 2×10⁶ CFU. FIG. 2 b shows that the SCID mice succumbed to theinfection with all three mycobacterial strains. However, the SCID micesuccumbed to an M. tuberculosis H37Rv intravenous infection within 2 to5 weeks, in a dose dependent manner. In the same time frame, the SCIDmice did not succumb to infection with M. tuberculosis H37Rv ΔRD1 untilweek 7, and only then, with the high dose of 2×10⁶ CFU. Mice receiving2×10³ CFU M. tuberculosis H37Rv ΔRD1 survived longer than 14 weeks postinfection, the survival rate of which coincided with the mice receiving2×10⁶ CFU of M. bovis BCG. Thus, these experiments established that M.tuberculosis H37Rv ΔRD1 was significantly more attenuated than itsparent, but not as attenuated as BCG-Pasteur in the immunocompromisedmice.

Colonial morphotypes of M. tuberculosis H37Rv ΔRD1. The M. tuberculosisH37Rv ΔRD1 mutant was generated independently three times from thesingle crossover construct (mc²4000) and upon subculturing, consistentlyyielded a 20 to 50% mixture of two colonial morphotypes on Middlebrookmedium without Tween 80 (FIG. 3 a). One morphotype was a smooth (S)phenotype that was flat and corded (like the parental M. tuberculosisH37Rv strain) and the second was a rough and raised (R) phenotype.Repeated subculturing of either the R or S colonies continued to yieldboth colonial morphotypes, but with a distribution of approximately 80%smooth and 20% rough colonies. The distinction of these two types ofmorphology could be noted even when the colonies were less than twoweeks old as the rough colonies were constricted and elevated with onlya small portion of the base of the colony attached to the agar, whilethe smooth colonies tends to be flattened and spread out. When coloniesgrew older, e.g. 6 weeks old, the rough colonies remained moreconstricted compared to those of smooth colonies. The rough coloniesexhibited large folds on the surface (FIG. 3 f, g), as compared to thoseof the smooth colonies that exhibited small wrinkles (FIG. 3 e).

Interestingly, in 1929, Petroff et al. reported a similar property foran early-derived BCG strain (Petroff et al., 1929) and proposed that theattenuation phenotype of BCG was not stable. Calmette disputed that theavirulent phenotype reverted and postulated that Petroff et al. hadacquired a contaminating virulent strain. Southern analysis of R and Scolonies revealed each morphotype has the same RD1-deleted genotype(FIG. 3 d). Furthermore, complementation of M. tuberculosis H37Rv ΔRD1with the RD1 region restored the mutant phenotype back to the homogenousparental S phenotype (FIG. 3 a-c). These results suggest that thevariable morphotypes resulted directly from the RD1 deletion thusdissociating a direct correlation of virulence with morphotype.

The M. tuberculosis H37Rv ΔRD1 is highly attenuated in immunocompetentBALB/c mice. To further assess the pathogenicity, survival, growthkinetics, and the histopathological analysis of the M. tuberculosisH37Rv ΔRD1 mutant, we compared the parental M. tuberculosis H37Rv toBCG-Pasteur strains in BALB/c mice. In survival studies, greater than50% BALB/c mice had died at 14 weeks post i.v. infection with 2×10⁶ CFUsof M. tuberculoisis H37Rv strain (FIG. 4 a). In contrast, all miceinfected with a similar dose of either BCG or M. tuberculosis H37Rv ΔRD1survived for longer than 22 weeks. These results were substantiated in aseparate experiment in which a group of 11 BALB/c mice were infectedwith 1×10⁵ CFU of M. tuberculosis H37Rv ΔRD1 and 9 of 11 mice (81%)survived greater than 9 months post-infection (data not shown). WhileBCG and M. tuberculosis H37Rv ΔRD1 showed similar survival results, thegrowth relative kinetics in mouse organs differed substantially. BCGgrew in a limited fashion in lungs, liver and spleen in BALB/c mice andwas cleared to undetectable levels by week 12 (FIG. 4 b-d). In contrast,the M. tuberculosis H37Rv ΔRD1 strain grew in a fashionindistinguishable from the parental M. tuberculosis H37Rv in all mouseorgans for the first 8 weeks. Thereafter, mice infected with theparental M. tuberculosis failed to contain the infection leading tomortality. Strikingly, mice infected with the M. tuberculosis H37Rv ΔRD1showed a definite control over infection resulting in significantlyprolonged survival of mice (FIG. 4 b-d).

The differing survival data of the three strains was clearlysubstantiated by histopathological analysis. M. tuberculosis H37Rv ΔRD1caused less severe organ damage in the lung, liver and spleen than thehighly virulent parent strain M. tuberculosis H37Rv. M. bovis BCG wasthe least virulent of the three strains. Based on histopathologicalevaluation, the mortality in mice infected with the wild type M.tuberculosis H37Rv (documented above and in FIG. 4 a) was caused byworsening pneumonia, hepatitis and splenitis (FIG. 5 a-c). Mice examinedat 14 weeks post-infection had developed severe lobar granulomatouspneumonia. Acid fast staining demonstrated large numbers of M.tuberculosis H37Rv, often in clumps, throughout the lung. The livers andspleens showed a severe diffuse granulomatous inflammation.

Histopathological examination further demonstrated that M. tuberculosisH37Rv ΔRD1 was attenuated in virulence compared to the parent strain M.tuberculosis H37Rv (FIG. 5 d-f). In contrast to the rapidly progressiveinfection with the parent strain M. tuberculosis H37Rv, the lung lesionscaused by M. tuberculosis H37Rv ΔRD1 were maximal in mice examined at 8weeks post-infection. Consolidating granulomatous pneumonia involved anestimated 25-30% of the lung in these mice. Numerous organisms weredemonstrated by acid fast staining. The pneumonia subsequently underwentpartial resolution. By 14 weeks, and again at 22 weeks post-infection,the lungs showed peribronchial and perivascular inflammatory cellaccumulations and focal, generally non-confluent, granulomas now with aprominent lymphocytic infiltration. The numbers of acid fast organismswere reduced. Liver lesions consisted of low numbers of scatteredgranulomas. Spleens were smaller, with persistent granulomas in the redpulp.

Mice infected with M. bovis BCG showed mild lesions in the lung, liverand spleen at all time points (FIG. 5 g-i). At longer time intervalspost-infection the lesions were fewer in number, smaller with prominentlymphocytic infiltrations. At 14 weeks post-infection, M. bovis BCG wasbelow the level of detection by acid fast staining. In summary, whereasM. tuberculosis H37Rv ΔRD1 initially grew in a manner similar to theparental M. tuberculosis H37Rv, this RD1 mutant was limited in theextent of spread of infection, particularly in the lung. This contrastedto the extensive and severe damage caused by the parent strain. Thesubsequent resolving granulomas, localization of the lesions and changesin the composition of the inflammatory cell infiltrations suggested thatthe mice mounted an effective immune response to combat M. tuberculosisH37Rv ΔRD1 infection and thereby reduced the numbers of viableorganisms.

M. tuberculosis H37Rv ΔRD1 protects mice against aerosolized M.tuberculosis challenge. To test the potential of M. tuberculosis H37RvΔRD1 to immunize mice and protect against tuberculous challenge, we usedthe model of subcutaneous immunization followed by aerosol challengewith virulent M. tuberculosis. Our initial studies in C57BL/6 micemonitored the growth the M. tuberculosis H37Rv ΔRD1 strain over an84-day period. Groups of mice (5 mice per group) were vaccinatedsubcutaneously (sc) either once or twice, 6 weeks apart, with 10⁶ CFU M.tuberculosis H37Rv ΔRD1 organisms. Additional mice were infectedintravenously (iv) with the same dose of the RD1-deleted strain in orderto examine the pathogenicity in C57BL/6 mice.

As seen in Table 1, M. tuberculosis H37Rv ΔRD1 persisted in the lungs,liver, and spleen for 3 months at moderate levels of infection but theorganisms failed to grow substantially in the lungs and spleens of micethat had been inoculated iv. In contrast, reduced persistence anddecreased concentrations of M. tuberculosis H37Rv ΔRD1 organisms weredetected in organ homogenates prepared from mice that had beenvaccinated sc. For the groups of mice that had been immunized with onlyone dose sc., low levels of M. tuberculosis H37Rv ΔRD1 bacilli wererecovered from the spleen after 28 and 56 days post-vaccination;however, no splenic mycobacteria were detected 84 days after the sc.injection. Importantly, the concentration of M. tuberculosis H37Rv ΔRD1organisms in the lungs after the sc. immunizations was below thethreshold of detection (<100 CFUs per organ) for the CFU assay at nearlyall time points during the three month study.

TABLE 1 Growth kinetics in C57BL/6 mice. Lung (Log CFU) Spleen (Log CFU)Weeks i.v. s.c. s.c. (2×) i.v. s.c. s.c. (2×) 4 5.86 ± <2 not done 5.73± 2.41 ± not done 0.10 0.05 0.26 8 5.79 ± <2 2.52 ± 5.37 ± 3.12 ± 3.62 ±0.07 0.34 0.04 0.40 0.29 12 5.61 ± <2 <2 5.40 ± <2 3.52 ± 0.09 0.05 0.22Mice were infected with 10⁶ M. tuberculosis H73Rv ΔRD1 by differentroutes. The data are presented as mean ± standard error of the mean.

Three months after the Sc. vaccinations with the ΔRD1 strain, groups ofmice were challenged aerogenically with a low dose (50 CFUs) of anacriflavin-resistant strain of M. tuberculosis Erdman. The use of adrug-resistant challenge strain permitted the differentiation of thechallenge organisms from the sensitive vaccine population. As controls,other groups of mice were immunized Sc. with 10⁶ CFUs of BCG Pasteur.The protective responses induced by the M. tuberculosis H37Rv ΔRD1vaccination were evaluated by assessing the relative growth of theacriflavin-resistant challenge organisms in naive, BCG vaccinated, andM. tuberculosis H37Rv ΔRD1 immunized mice and by comparing the relativepost-challenge lung pathology in the experimental groups and the naivecontrols. As seen in Table 2, the growth of the drug-resistant challengeorganisms was substantially lower in the lungs of animals vaccinatedwith BCG or the M. tuberculosis H37Rv ΔRD1 vaccine. Significantreductions in the lung CFU values in the vaccinated animals (relative tonaive controls) could be detected both 28 and 56 days after thechallenge. Dissemination to the spleen was also significantly limited inall of the vaccination groups with the most substantial differences(−1.4 log₁₀ CFUs compared to the naives) being detected during the firstmonth post-challenge. While significant differences in the growth of themycobacterial challenge was identified between unvaccinated andvaccinated mice, the rate of proliferation of the acriflavin-resistantchallenge strain in all the experimental groups (BCG sc or M.tuberculosis H37Rv ΔRD1 or 2 doses sc) was nearly identical and notstatistically different.

TABLE 2 M. tuberculosis ΔRD1 and BCG protect C57BL/6 mice from areosolchallenge with M. tuberculosis Erdman Lung (Log CFU) Spleen (Log CFU)Day 28 Day 56 Day 28 Day 56 Naive 4.77 ± 0.06 4.11 ± 0.05 3.57 ± 0.213.20 ± 0.16 BCG (1×) 3.96 ± 0.20 3.80 ± 0.08 2.18 ± 0.18 2.48 ± 0.23ΔRD1 (1×) 3.97 ± 0.39 3.71 ± 0.06 2.12 ± 0.12 2.60 ± 0.25 ΔRD1 (2×) 3.96± 0.15 3.66 ± 0.09 2.21 ± 0.15 2.22 ± 0.16 Immunizations were performedsubcutaneously once (1×) or twice (2×) with 2 × 10⁶ CFUs of thevaccinating strains. Three months later, vaccinated animals wereaerogenically challenged with 50 CFUs/mouse of acriflavin resistant M.tuberculosis Erdman. The growth of the bacterial challenge was monitored28 and 56 days post infection by plating on Middlebrook 7H11 platescontaining 20 μg/ml acriflavin and using procedures previously described(Delogu et al., 2002).Discussion

The M. tuberculosis H37Rv ΔRD1 mutant strain shares significantproperties with BCG including: 1) a significant attenuation of virulencein mice, 2) the ability to generate variable colonial morphotypes, and3) the ability to protect mice against aerogenic tuberculosis challenge.These properties, and the observation that RD1 is the only deletioncommon to all BCG substrains, makes it likely that the RD1 deletion isthe primary attenuating mutation. It remains to be determined if asingle gene or a number of genes in this region causes the attenuatedphenotype. The variable colonial morphotype switch does suggest that aprotein regulating cell wall biogenesis is affected. Notably, definedmutations affecting the cyclopropanation of mycolic acids (Glickman etal., 2000) or the synthesis or export of phthiocerol dimycoseroate (Coxet al., 1999) have been found to correlate with decreased virulence andaltered colony morphotypes in M. tuberculosis and thus representattractive candidate genes that might be regulated by an RD1-encodedgene. The M. tuberculosis ΔRD1 mutant provides a precisely definedbackground strain by which to determine virulence and colony morphologyrelated genes.

BCG is currently the only antituberculous vaccine available for use inhumans. In many animal models, BCG has been shown to induce protectiveimmunity against M. tuberculosis challenge (Opie and Freund, 1937;Hubbard et al., 1992; Baldwin et al., 1998) and in addition, hasdemonstrated protection against the most severe and fatal form of TB inchildren (Rodrigues et al., 1991). However, BCG has shown variableefficacy in protecting adults from pulmonary TB (Tuberculosis PreventionTrial, 1980; Hart and Sutherland, 1977; Bloom and Fine, 1994). Due tothe uncertain efficacy of BCG, particularly in TB-endemic countries, thedevelopment of improved tuberculosis vaccines has become aninternational research priority.

Our challenge studies have demonstrated that the protective immuneresponses elicited by immunization with M. tuberculosis H37Rv ΔRD1 inmice are at least as strong as the protective responses induced byvaccination with BCG. The M. tuberculosis H37Rv ΔRD1 mutant also retainsthe BCG-associated property of limited spread to the lung followingsubcutaneous immunization. Restricted dissemination of the ΔRD1 mutantto the lung suggests it should have a favorable overall safety profile.Also, the unmarked mutant of M. tuberculosis H37Rv ΔRD1 provides asingle deletion strain whereby other attenuating mutations can bereadily engineered. Since the risk of reversion to wild-type virulencedecreases substantially with each additional attenuating mutation, M.tuberculosis mutants harboring deletions in two or three separategenetic loci should provide a much safer vaccine for long term use.

M. tuberculosis mutants with RD1 deletions represent attractivecandidates as novel vaccines for TB prevention. These mutants, derivedfrom a single mutagenic event from the parental M. tuberculosis strain,replicate more efficiently in vivo than BCG, especially early ininfection. This enhanced rate of proliferation for the RD1-deletedstrains, relative to BCG, may lead to the induction of increasedprotective immunity in humans, after vaccination with M. tuberculosisH37Rv ΔRD1. Moreover, they could also be more immunogenic as there existat least 129 ORFs present in M. tuberculosis H37Rv that are absent fromM. bovis (Behr et al., 1999). Since some of these ORFs are likely toencode regulatory proteins affecting the expression of other genes,there could be hundreds of antigens expressed in M.tuberculosis-infected cells that are absent from BCG-infected cells.Thus, RD1 deletion mutants constructed from human tubercle bacilli couldprotect humans against disease substantially better than BCG.

EXAMPLE 2 Vitamin Auxotrophs of Mycobacterium Tuberculosis areAttenuated and Protect Against Tuberculosis

This example describes experimental methods and results that establishthat deleting genes that control vitamin production in a virulent M.tuberculosis causes the M. tuberculosis to become avirulent and sustainan infection in mammals, and protect the mammal against challenge with avirulent M. tuberculosis.

Given the importance of NAD and nicotinamide (vitamin B3) andpantothenate (vitamin B5) as cofactors involved in carbon utilization,energy transduction (Abiko, 1975; Jackowski, 1996) and the biosynthesisof the complex lipid cell wall of M. tuberculosis, we hypothesized thatmutations in the biosynthetic pathways for NAD and pantothenate couldlead to the generation of mutant strains that retain a limited abilityto replicate and subsequently get cleared within the host tissues. In M.tuberculosis, the nadABC operon controls the de novo biosynthesis ofNAD. Similarly, the panC and panD genes that are organized in an operoncontrol the rate-limiting step in the de novo biosynthesis ofpantothenate. We constructed deletion mutants of M. tuberculosis in thenadBC and panCD genes using specialized transduction, as described inExample 1. The mutant strains mc²3122 (ΔnadBC) and mc²6001 (ΔpanCD) wereauxotrophic for nicotinamide and pantothenate respectively. The in vitroreversion frequencies of the respective mutations were found to be lessthan 10⁻¹⁰ events per generation.

The safety and attenuation of ΔnadBC and ΔpanCD auxotrophic mutants wereassessed by infection of immune-compromised SCID mice. SCID miceinfected with wild-type M. tuberculosis and the ΔnadBC mutant succumbedto infection in about 5 weeks (data not shown). This result clearlyindicates that in the absence of T-cell immunity, intermediates of NADbiosynthetic pathway, such as nicotinamide, are readily available in themacrophages to support the growth of the ΔnadBC mutant. In contrast allmice infected with the ΔpanCD mutant survived longer than 30 weeks,demonstrating the severe attenuation of this mutant strain. The fullvirulence phenotype was restored when the panCD wild type alleles wereintegrated into the chromosome of the ΔpanCD mutant in single copy,suggesting the observed attenuation in ΔpanCD to be due to therequirement of pantothenate for growth and not due to polar effects ofthe mutation on downstream genes. SCID mice infected with the same doseof conventional BCG-Pasteur vaccine strain succumbed to infection within80 days (FIG. 6A) in accordance with earlier reports (Guleria, 1996).Enumeration of bacterial burdens in SCID mice infected with wild type M.tuberculosis H37Rv and the complementing strain (panCD in single copyintegrated into the chromosome) showed a rapid increase in bacterialnumbers in spleen, liver and lung before they succumbed to infection. Incontrast, mice infected with ΔpanCD mutant, showed an initial drop inbacterial numbers in spleen and liver followed by a steady increase toreach 10⁸ in the lungs at 160 days, at which time all mice were stillalive (FIG. 6B).

Having demonstrated the significant attenuation of ΔpanCD mutant, wesought to address the in vivo growth characteristics of this mutant inimmune-competent BALB/c mice. All BALB/c mice infected with H37Rvsuccumbed to infection by day 25 with a MST of 22 days. Similarly, miceinfected with the panCD-complemented strain were highly virulent with100% mortality between 3-8 weeks post-infection similar to the wild typestrain, with a MST of 28 days. In contrast, all mice infected withΔpanCD mutant survived for over 33 weeks demonstrating the severeattenuation phenotype of this mutant in immune-competent mice (FIG. 7A).Interestingly, bacterial enumeration at three weeks post infectionshowed 1 log increase in the ΔpanCD numbers in lungs followed by a stateof persistence with the onset of adaptive immune response. This growthcharacteristic was observed only in the lung but not in spleen or liver(FIG. 7B,C). A desirable trait of an effective live attenuated vaccinestrain is its ability to grow within the host in a limited fashion inorder to produce in vivo all the important protective antigens(McKenney, 1999; McKenny, 2000; Kanai, 1955). The ΔpanCD mutant exhibitsthis characteristic in the lung, which is the primary site of infectionin humans and does not get cleared over a prolonged period in all thethree organs. The earlier auxotrophs of M. tuberculosis failed to growin any of the organs and hence failed to adequately protect againstexperimental challenge in guinea pigs (Jackson, 1999), or mice.

The ability of the ΔpanCD mutant to exhibit limited growth in the lunguntil the onset of adaptive immune response suggests that anunidentified putative pantothenate permease is able to transport thisnutrient into resting macrophages, as in the SCID mice. Asodfum-dependent pantothenate permease actively transports pantothenateinto the cell of Escherichia coli (Vallari and Rock, 1985; Jackowski andAlix, 1990), Plasmodium falciparum (Saliba and Kirk, 2001) and mammals.Subsequent activation of macrophages leads to restricted supply of thisnutrient within the phagosome resulting in growth arrest of the mutant.Pantothenic acid or its derivatives have been reported to conferresistance to radiation and oxidative stress by virtue of their role inbiosynthesis of CoA and also by indirectly increasing the cellularsupply of glutamate, a precursor of glutathione (Slyshenkov, 1995).Pantothenate kinase (PanK) mutants of Drosophila display membranedefects and improper mitosis and meiosis due to decreased phospholipidbiosynthesis (Afshar et al., 2001). The disruption of de novopantothenate biosynthesis causes an increased susceptibility of theΔpanCD mutant to reactive oxygen and nitrogen intermediates that arereleased within activated macrophages.

Having observed the ΔnadBC mutant to be non-attenuated in SCID mice, wechose to study the in vivo growth kinetics of this mutant in the moreresistant C57BL/6 mice background. During the first three weeks ofinfection, the number of wild type and mutant bacteria recovered fromall three organs showed little or no difference. Their numbers graduallyincreased in the lungs to reach 10⁶. However, with the onset of adaptiveimmune response at three weeks, when the growth of bacteria in the lungsof mice infected with H37Rv became constant and tightly controlled,bacterial load in the lungs of mice infected with ΔnadBC mutant showed aconstant tendency for clearance to reach more than 1.5 log drop in thebacterial numbers compared to mice infected with wild type strain (FIG.8A). This difference was preserved up to 24 weeks following infection.

The reduced ability of the ΔnadBC mutant to sustain an infection wasaccompanied by attenuated virulence clearly seen from the survivalexperiment (FIG. 8C). While all mice infected with the wild type strainsuccumbed to infection between day 90 and 179 (MST 116 days) all miceinfected with the ΔnadBC mutant (n=12) remain alive for a period of morethan 8 months (FIG. 8C).

Our observation of the attenuation phenotype of ΔnadBC mutant becameobvious only after the onset of immune response, suggesting that oncethe macrophages become activated, they restrict the amount of availableNAD or NAD intermediates causing a restricted growth of the mutantstrain. This would be in agreement with the recently reportedobservations that a significant part of antimicrobial function of themacrophages could be attributed to the IFN-γ promoted enhancedexpression of indolamine 2-oxygenase (IDO), the inducible enzymecontrolling L-tryptophan catabolic pathway causing an almost completedepletion of L-tryptophan pool. The enhanced catabolism of L-tryptophanleads to increased de novo biosynthesis of NAD needed to protect thecells from the free radicals formed as a result of macrophageactivation. Recently, several studies have demonstrated the involvementof the tryptophan catabolism in the antimicrobial mechanisms of theactivated macrophages. Induction of IDO was found responsible for theinhibition of intracellular growth of Toxoplasma, Leishmania, Legionellaand Chlamydia. The restricted intracellular growth of ΔnadBC mutantcould be explained with the very little amount of free NAD or NADintermediates available within the activated macrophages.

Having established the safety and persistence of ΔpanCD and ΔnadBC inimmunocompetent mice, the protective efficacy of these mutants wereevaluated using an aerosol challenge model with virulent M.tuberculosis, using the methods described in Example 1. The aerosolroute of infection was chosen, as this is the natural route of infectionin humans. To assess the capacity of the auxotrophic vaccines torestrict growth of virulent M. tuberculosis in the organs of infectedmice, bacterial numbers were enumerated one month post-infection in lungand spleen. See Table 3. In the unimmunized controls, bacterial numbersrose rapidly in the spleen and lungs, in contrast mice infected with asingle dose of ΔpanCD displayed significant reduction in bacterialnumbers in the spleen and lung (p<0.05, in comparison to unimmunizedcontrols). Mice given two doses of ΔpanCD displayed a statisticallysignificant reduction in the bacterial numbers to 4.5 log units in thelung (p<0.01) and 3.7 log units in the spleen (p<0.05). Mice vaccinatedwith BCG showed comparable reduction in bacterial burden in the lung andspleen to 3.3 log units and 4.7 log units respectively (p<0.01). Miceimmunized with one or two doses of ΔnadBC mutant conferred statisticallysignificant protection (p<0.01 in comparison to unimmunized group) thatis comparable to the protection afforded by BCG vaccination.Interestingly, mice immunized with the ΔnadBC mutant showed nodetectable CFUs in the spleen suggesting that the vaccination completelyprevented the hematogenous spread of wild type M. tuberculosis followingaerosol challenge.

TABLE 3 Experimental Group Lung CFUs (log₁₀) Spleen CFUs (log₁₀) A Naive4.05 ± 0.21 3.94 ± 0.21 ΔnadBC (1 × sc) 3.37 ± 0.40** <2** ΔnadBC (2 ×sc)  3.6 ± 0.35** <2** BCG (1 × sc) 3.46 ± 0.19** <2** B Naive 5.56 ±0.05 4.35 ± 0.21 ΔpanCD (1 × sc) 4.99 ± 0.17 (−0.57)* 3.65 ± 0.15(−0.70)* ΔpanCD (2 × sc) 4.55 ± 0.09 (−1.01)** 3.73 ± 0.21 (−0.62)* BCG(1 × sc) 4.71 ± 0.21 (−0.85)** 3.35 ± 0.20 (−1.00)** p < 0.05 comparedto naïve, **p < 0.01 compared to naïve

Table 3. The attenuated M. tuberculosis ΔnadBC and ΔpanCD mutantsprotect against aerogenic challenge with M. tuberculosis Erdman. Groupsof C57BL/6 mice (5 mice per group) were vaccinated subcutaneously eitheronce or twice (6 weeks apart) with 10⁶ CFUs of mutant strains. Controlmice were vaccinated subcutaneously with 10⁶ CFUs of BCG-Pasteur. Threemonths after the initial immunization with either ΔnadBC or ΔpanCDmutant or BCG, the mice were aerogenically challenged withapproximatedly 100 CFUs of acriflavin-resistant M. tuberculosis Erdman(Ac^(r)MTB) strain as described earlier (Collins, 1985) After 28 days,the challenged mice were sacrificed, and the lungs and spleens ofindividual mice were removed aseptically and homogenized separately in 5ml of Tween 80-saline using a Seward stomacher 80 blender (Tekmar,Cincinnati, Ohio). The homogenates were diluted serially in Tween 80saline and plated on Middlebrook 7H11 agar with or without appropriatesupplements as required. Samples from the BCG-vaccinated controls wereplated on 7H11 agar containing 2 mg of thiophenecarboxylic acidhydrazide (Sigma Chemical Co., St Louis, Mo.) per ml to inhibit growthof any residual BCG. The CFU results were evaluated using the one-wayANOVA analysis of the Graph Pad InStat program. The numbers inparanthesis represent the differences between naïve and vaccinated organCFUs.

In order to test the ability of the auxotrophic mutants to confer longlasting immunity, mice were challenged 7 months after an initialsubcutaneous immunization with the ΔnadBC mutant. See Table 4. Miceimmunized with ΔnadBC displayed significantly reduced numbers of thechallenge organism in the lungs and no detectable numbers in the spleencomparable to the numbers seen in the BCG vaccinated mice. The resultssuggest that the ΔnadBC vaccine strain is able to persist within themouse organs sufficiently long to mount a long lasting immunity tocontrol subsequent infection.

TABLE 4 Experimental Group Lung CFUs (log₁₀) Spleen CFUs (log₁₀) Naive4.61 ± 0.07 4.07 ± 0.20 BCG 4.00 ± 0.13*  2 NAD (1 × iv) 3.28 ± 0.15**<2 NAD (2 × iv) 2.95 ± 0.14** <2 NAD (1 × sc) 4.05 ± 0.12* <2 NAD (2 ×sc) 3.94 ± 0.13* <2 *P < 0.05; **P < 0.01 by Dunnett's MultipleComparison Test

Table 4. Immunizations with the ΔnadBC mutant confer long-termprotection against an aerosol challenge. Groups of C57BL/6 mice (5 miceper group) were vaccinated subcutaneously or intravenously either onceor twice (6 weeks apart) with 10⁶ CFUs of ΔnadBC mutant. Control micewere vaccinated subcutaneously with 10⁶ CFUs of BCG-Pasteur. Sevenmonths after the initial immunization with either ΔnadBC mutant or BCG,the mice were aerogenically challenged with approximately 50 CFUs ofacriflavin-resistant M. tuberculosis Erdman (Ac^(r)MTB) strain and thebacterial numbers at 28 days post challenge enumerated as described inTable 1.

To the best of our knowledge this is the first report of any M.tuberculosis auxotrophic vaccines administered subcutaneously to conferprotection comparable to the conventional BCG vaccine strain in a mousemodel of infection. Mice vaccinated with the ΔpanCD and ΔnadBC survivedfor over one year following the aerosol challenge indicating theprotection and safety of these vaccine strains.

EXAMPLE 3 A Pantothenate Auxotroph of Mycobacterium Tuberculosis isHighly Attenuated and Protects Mice Against Tuberculosis

This Example is published as Sambandamurthy et al., 2002. Examplesummary.

With the advent of HIV and the widespread emergence of drug resistantstrains of Mycobacterium tuberculosis, newer control strategies in theform of a better vaccine could decrease the global incidence oftuberculosis. A desirable trait in an effective live attenuated vaccinestrain is its ability to persist within the host in a limited fashion inorder to produce important protective antigens in vivo (Kanai andYanagisawa, 1955; McKenney et al., 1999). Rationally attenuated M.tuberculosis vaccine candidates have been constructed by deleting genesrequired for growth in mice (Jackson et al., 1999; Hondalus et al.,2000; Smith et al., 2001). These candidate vaccines failed to elicitadequate protective immunity in animal models, due to their inability topersist sufficiently long within the host tissues. Here we report thatan auxotrophic mutant of M. tuberculosis defective in the de novobiosynthesis of pantothenic acid (vitamin B5) is highly attenuated inimmunocompromised SCID mice and in immunocompetent BALB/c mice. SCIDmice infected with the pantothenate auxotroph survived significantlylonger than mice infected with either BCG vaccine or virulent M.tuberculosis (250 days, vs. 77 days, vs. 35 days). Subcutaneousimmunization with this auxotroph conferred protection in C57BL/6J miceagainst an aerosol challenge with virulent M. tuberculosis, which wascomparable to that afforded by BCG vaccination. Our findings highlightthe importance of de novo pantothenate biosynthesis in limiting theintracellular survival and pathogenesis of M. tuberculosis withoutreducing its immunogenicity in vaccinated mice.

Materials and Methods

Media and Strains. M. tuberculosis H37Rv, M. tuberculosis Erdman and M.bovis BCG Pasteur were obtained from the Trudeau Culture Collection(Saranac Lake, N.Y.) and cultured in Middlebrook 7H9 broth and 7H11 agarsupplemented with 10% OADC, 0.5% glycerol, and 0.05% Tween 80. Whenrequired, pantothenate (24 μg/ml), hygromycin (50 μg/ml) or kanamycin(25 μg/ml) was added. Stock strains were grown in Middlebrook 7H9 brothin roller bottles and harvested in mid-logarithmic growth phase, beforebeing stored in 1 ml vials at −70° C. until required.

Disruption of panCD genes in M. tuberculosis. Specialized transductionwas employed to disrupt the chromosomal copy of the panCD genes asdescribed (U.S. Pat. No. 6,271,034). Briefly, the 823 bp region upstreamto the panC gene was amplified using primers Pan1(5′-GTGCAGCGCCATCTCTCA-3′)(SEQ ID NO:9)and Pan2(5′-GTTCACCGGGATGGAACG-3′)(SEQ ID NO:10). A 716 bp region downstream tothe panD gene was amplified using primers Pan3(5′-CCCGGCTCGGTGTGGGAT-3′) (SEQ ID NO:11) and Pan4(5′-GCGCGGTATGCCCGGTAG-3′)(SEQ ID NO:12). PCR products were cloned withthe TOPO TA cloning kit (Invitrogen, Calif.), and sequenced. PCRproducts were subsequently cloned into pJSC347, flanking a hygromycincassette to create pSKV1. PacI digested pSKV1 was ligated into thetemperature-sensitive mycobacteriophage phAE159 derived from TM4 andtransduced as described earlier (Glickman et al., 2000; Raman et al.,2001). Genomic DNAs from hygromycin-resistant and pantothenate-requiringcolonies were digested with BssHII, and probed with a 716 bp downstreamregion, flanking the M. tuberculosis panCD operon to confirm thedeletion. For complementation, the M. tuberculosis panCD operon wasamplified by PCR from genomic DNA with its putative promoter, clonedwith TA cloning kit, sequenced, and subcloned into pMV306kan, asite-specific integrating mycobacterial vector.

Animal infections. C57BL/6, BALB/cJ and BALB/c SCID mice (6-8 weeks old)were purchased from Jackson Laboratories and were infected intravenouslythrough the lateral tail vein. For time-to-death assays, BALB/c SCIDmice were infected intravenously with 1×10² CFU of M. tuberculosisH37Rv, 1×10² CFU of panCD-complemented strain, 1×10⁵ CFU of ΔpanCDmutant, or 1×10⁵ CFU of M. bovis BCG-P. For mouse organ CFU assays,BALB/cJ mice were infected with 1×10⁶ CFU of M. tuberculosis H37Rv orthe panCD-complemented strain or the ΔpanCD mutant. At appropriate timepoints, groups of 4-5 mice were sacrificed and the selected organs werehomogenized separately in PBS/0.05% Tween 80, and colonies wereenumerated on 7H11 plates grown at 37° C. for 3-4 weeks (see McKinney etal., 2000). Pathological examination was performed on tissues fixed in10% buffered formalin. The CFU results were evaluated using the one-wayANOVA analysis of the Graph Pad InStat program. All animals weremaintained in accordance with protocols approved by the Albert EinsteinCollege of Medicine Institutional Animal Care and Use Committee.

Vaccination Studies. Groups of C57BL/6 mice (5 mice per group) werevaccinated subcutaneously either once or twice (6 weeks apart) with1×10⁶ CFU of the ΔpanCD mutant strain. Control mice were vaccinatedsubcutaneously with 1×10⁶ CFU of M. bovis BCG-P. Three months after theinitial immunization with either the ΔpanCD mutant or BCG, the mice wereaerogenically challenged with approximately 50-100 CFU of M.tuberculosis Erdman strain as described earlier. At 28 days followingaerosol challenge, the challenged mice were sacrificed; the lungs andspleens of individual mice were removed aseptically and homogenizedseparately in 5 ml of Tween 80-saline using a Seward Stomacher 80blender (Tekmar, Cincinnati, Ohio). The homogenates were dilutedserially in Tween 80 saline and plated on Middlebrook 7H11 agar with orwithout appropriate supplements as required. Samples from theBCG-vaccinated controls were plated on 7H11 agar containing 2 mg/ml ofthiophene-2-carboxylic acid hydrazide (Sigma) to inhibit growth of anyresidual BCG.

Results and Discussion

Lipid biosynthesis and metabolism have been shown to play a pivotal rolein the intracellular replication and persistence of M. tuberculosis (Coxet al., 1999; Camacho et al., 1999; Glickman et al., 2000; De Voss etal., 2000; Manca et al., 2001; McKinney et al., 2000). Therefore, wesought to globally impair the ability of this bacterium to synthesizelipids. Pantothenic acid (vitamin B5) is an essential molecule requiredfor the synthesis of coenzyme A (CoA) and acyl carrier protein (ACP),that play important roles as acyl group carriers in fatty acidmetabolism, the tricarboxylic acid cycle, biosynthesis of polyketidesand several other reactions associated with intermediary metabolism(Jackowski, 1996). Bacteria, plants and fungi synthesize pantothenatefrom amino acid intermediates, whereas it is a nutritional requirementin higher animals (FIG. 9 a).

We constructed a double deletion mutant of M. tuberculosis in the panCand panD genes that are involved in the de novo biosynthesis ofpantothenate (FIG. 9 b,c). The ΔpanCD mutant was found to be auxotrophicfor pantothenate with no detectable reversion to prototrophy when 1×10¹⁰cells were plated on minimal medium. The growth rate of the mutant wasidentical to wild type H37Rv in broth cultures in the presence ofexogenous pantothenate (data not shown). The attenuation of the ΔpanCDmutant was assessed by infection of immunocompromised SCID mice. SCIDmice infected intravenously with H37Rv succumbed to the resultinginfection in about 5 weeks. In contrast, all mice infected with theΔpanCD mutant survived for more than 36 weeks (average, 253 days) (FIG.10 a). This attenuation is due to pantothenate auxotrophy as the fullvirulence phenotype was restored when the panCD wild type genes wereintegrated into the chromosome of the ΔpanCD mutant in single copy.Enumeration of bacterial burdens in SCID mice infected with H37Rv andthe ΔpanCD-complemented strain showed a rapid increase in bacterialnumbers in the spleen, liver and lung, until they succumbed toinfection. In contrast, mice infected with the ΔpanCD mutant showed aninitial drop in bacterial numbers in the spleen and liver followed by agradual increase in the number of viable bacteria, reaching 1×10⁶colony-forming units (CFU) by day 224 (FIG. 10 b). Notably, the CFUvalues increased to 1×10⁸ in the lungs of the infected mice. The abilityof ΔpanCD-infected SCID mice to survive despite a substantial bacterialburden in their lungs emphasizes the extent of attenuation in thismutant and compares with the phenotype observed with the M. tuberculosiswhiB3 and sigH mutants described recently (Steyn et al., 2000; Kaushal,2000). Notably, SCID mice infected with bacille Calmette-Guerin-Pasteur(BCG-P) strain succumbed to infection by 83 days (Weber et al., 2000) incontrast to the prolonged survival observed in ΔpanCD-infected mice.

Studies in immunocompetent mice further demonstrate the attenuation ofthe ΔpanCD mutant. Survival studies showed that BALB/c mice infectedwith H37Rv succumbed to infection by day 25 (average, 21 days) and miceinfected with an identical dose of the panCD-complemented strainsuccumbed to infection between days 21 to 53 (average, 37 days).Importantly, all mice infected with 1×10⁶ CFU of the ΔpanCD mutantsurvived 375 days, when the experiment was terminated (FIG. 10 c). At 3weeks post infection, in contrast to the H37Rv strain, BALB/c miceinfected with ΔpanCD mutant showed a 10-fold increase in bacterialnumbers in the lungs followed by a gradual decline in viable numbersover the next 38 weeks of infection (FIG. 10 d) and the bacterial burdengradually declined in the spleen and liver throughout the course ofinfection (FIG. 10 e). Histopathologic examination of the lungs frommice infected with either H37Rv or the ΔpanCb-complemented strain,showed severe, diffuse lobar granulomatous pneumonia (FIGS. 11 a,b). Thepneumonia affected more than 50% of the lung, and was pyogranulomatouswith marked necrosis in the advanced consolidated areas, particularly inthe lungs of mice challenged with H37Rv. Both of these strains causedsevere granulomatous splenitis and widespread granulomatous hepatitis.At 3 weeks post-infection with the ΔpanCD mutant, low to moderatenumbers of focal infiltrates of mononuclear cells scattered throughoutthe lung were seen (FIG. 11 c). The spleen was moderately enlarged withscattered granulomas. Similarly, the liver showed numerous focalgranulomas. At 24 weeks post-infection, consistent with the bacterialnumbers, histological examination of the lungs from mice infected withthe ΔpanCD mutant showed only occasional focal mild infiltrations,predominately lymphocytic (FIG. 11 d). The spleen showed only mildhistiocytic hyperplasia and there were fewer, focal, predominatelylymphocytic accumulations in the liver.

The mechanisms that allow the persistence of the ΔpanCD mutant bacteriafor over 8 months in the SCID mouse model remain unclear. We speculatethe functional role of an unidentified permease in transporting adequateamount of pantothenate in the ΔpanCD mutant that allows its persistencebut not the ability to cause disease. A pantothenate permease thattransports pantothenate have been described in Plasmodium falciparum andEscherichia coli (Saliba and Kirk, 2001; Jackowski and Alix, 1990). Inthe lungs of immunocompetent mice, an initial growth of the ΔpanCDmutant during the first 3 weeks is followed by a steady decline inbacterial numbers following the onset of an adaptive immune response.The intracellular lifestyle of M. tuberculosis poses significantchallenges to the bacterium in acquiring essential nutrients.Pantothenic acid or its derivatives have been shown to confer resistanceto oxidative stress (Slyshenkov et al., 1996) and lack of pantothenatebiosynthesis in the ΔpanCD mutant may render it more susceptible to suchadverse effects. Likewise, a pantothenate kinase (pank) mutant ofDrosophila was shown to display membrane defects and improper mitosisand meiosis due to decreased phospholipid biosynthesis (Afshar et al.,2001). Therefore, it is plausible that the pantothenate salvage pathwayis inadequate in restoring full virulence of the ΔpanCD mutant in theabsence of a functional de novo biosynthetic pathway.

As a test of vaccine potential, immunized mice were challenged withvirulent M. tuberculosis Erdman by the aerosol route (Collins, 1985).Following subcutaneous immunization, the ΔpanCD mutant could not bedetected in the spleens or lungs of mice at 8 and 12 weeks. In the naivecontrols, the bacterial CFU values increased 10,000-fold in the lungduring the first month after challenge. Similarly, substantialdissemination and growth in the spleen was detected within one month ofthe challenge in naive controls. In contrast, mice immunized with singleor double doses of the ΔpanCD mutant displayed statistically significantreductions (P<0.05) in lung and spleen CFU values relative to naivecontrols. Mice vaccinated with BCG showed similar reduction in organbacterial burdens compared to the nonimmunized controls (FIG. 11 e,f).In these aerogenic challenge studies, no significant differences weredetected in the lung and spleen CFU values for mice vaccinated witheither the ΔpanCD mutant strain or with BCG. At 28 days after theaerogenic challenge with virulent M. tuberculosis, histopathologicalexamination of lungs of ΔpanCD immunized mice revealed a less severeinfection relative to the unvaccinated control mice. In controls, severebronchitis, moderate pneumonia, and spread of the infection to theadjacent lung parenchyma was observed. By comparison, the ΔpanCDvaccinated mice had milder bronchitis and smaller areas of mildinterstitial pneumonitis, with localized areas of granulomatouspneumonia in some mice. Importantly, no lung pathology was detected invaccinated mice at the time of challenge (data not shown). Two groups ofmice that were vaccinated with one or two doses of the ΔpanCD mutant andthen challenged with M. tuberculosis Erdman were active and healthy formore than one year following the virulent challenge. Histopathologicalanalysis of lung sections from these mice showed only mild inflammationand fibrosis despite the chronic infection.

By creating a M. tuberculosis strain that is defective in pantothenatebiosynthesis, we have taken a critical step in the rational developmentof an attenuated M. tuberculosis vaccine strain. We have shown that afunctional pantothenate biosynthetic pathway, which is required for thesynthesis of complex mycobacterial lipids, is essential for thevirulence of M. tuberculosis. Although the precise mechanism of thereduced virulence is unclear, it is reasonable to speculate that thiscould be due to reduced synthesis of toxic polyketides and secretedlipids or a general slow down of metabolism. Tubercle bacilli lackingthe two genes required to synthesize pantothenate failed to revert andwere highly attenuated and less virulent than BCG vaccine when tested inthe rigorous SCID mouse model of infection. Despite the reducedvirulence associated with the deletion of the panCD genes, these vitaminauxotrophs remain persistent in vivo as shown by their ability tosurvive for at least eight months in immunocompetent mice. Thepersistence of this mutant strain undoubtedly contributes to thesubstantial immunogenicity seen in the mouse tuberculous challengemodel. Overall, the ΔpanCD mutant has many of the characteristicsnecessary for a live vaccine candidate strain: it is attenuated by anon-reverting mutation and essentially avirulent while being persistentand immunogenic. Given the genetic differences between M. bovis and M.tuberculosis (Behr et al., 1999), one would predict that a rationallyattenuated M. tuberculosis strain would have a more relevant repertoireof species-specific antigens and thus should elicit, in humans, moreeffective protective immune responses against tuberculous infectionsthan BCG.

EXAMPLE 4 The Primary Mechanism of Attenuation of BCG is a Loss ofInvasiveness Due to Host Cell Lysis

Example Summary

Tuberculosis remains a leading cause of death worldwide, despite theavailability of effective chemotherapy and a vaccine. BCG, thetuberculosis vaccine, is an attenuated mutant of M. bovis that wasisolated following serial subcultivations, yet the basis for thisattenuation has never been elucidated. A single region (RD1), deleted inall BCG substrains, was deleted from virulent M. bovis and M.tuberculosis strains and the resulting three ΔRD1 mutants weresignificantly attenuated for virulence in both immunocompromised andimmunocompetent mice. Like BCG, M. tuberculosis ΔRD1 mutants protectmice against acrosolized M. tuberculosis challenge and these mutantsalso consistently display altered colonial morphotypes. Interestingly,the ΔRD1 mutants failed to cause necrosis, via lysis, of pneumocytes, aphenotype that had been previously used to distinguish virulent M.tuberculosis from BCG. We conclude that the primary attenuatingmechanism of BCG is the loss of cytolytic activity, resulting in reducedinvasiveness.

Introduction

BCG (bacille Calmette and Guerin), was first isolated from M. bovisfollowing serial subculturing of M. bovis in 1908 (Calmette and Guerin,1909). Drs. Calmette and Guerin set out to test the hypothesis that abovine tubercle bacillus could transmit pulmonary tuberculosis followingoral administration (Calmette and Guerin, 1905; Gheorghiu, 1996) anddeveloped a medium containing beef bile that enabled the preparation offine homogenous bacillary suspensions. After the 39th passage, thestrain was found to be unable to kill experimental animals (Calmette andGuerin, 1909). Between 1908 and 1921, the strain showed no reversion tovirulence after 230 passages on bile potato medium (Gheorghiu, 1996),which is consistent with the attenuating mutation being a deletionmutation. In the animal studies that followed, BCG was shown to beattenuated, but it also protected animals receiving a lethal challengeof virulent tubercle bacilli (Calmette and Guerin, 1920). BCG was firstused as a vaccine against tuberculosis in a child in 1921 (Weill-Halleand Turpin, 1925). From 1921 to 1927, BCG was shown to have protectiveefficacy against TB in a study on children (Id.; Calmette and Plotz,1929) and was adopted by the League of Nations in 1928 for widespreaduse in the prevention of tuberculosis. By the 1950's, after a series ofclinical trials, the WHO was encouraging widespread use of BCG vaccinethroughout the world (Fine and Rodrigues, 1990). Although an estimated 3billion doses have been used to vaccinate the human population againsttuberculosis; the mechanism that causes BCG's attenuation remainsunknown.

Mahairas et al.(1996) first compared the genomic sequences of BCG and M.bovis using subtractive hybridization and found that there were threeRegions of Difference (designated RD1, RD2, and RD3) present in thegenome of M. bovis, but missing in BCG. Behr et al. (Behr et al., 1999)and others (Gordon et al., 2001) later identified 16 large deletions,including RD1 to RD3, present in the BCG genome but absent in M.tuberculosis. Eleven of these 16 deletions were unique to M. bovis,while the remaining 5 deletions were unique to BCG. One of these 5deletions, designated RD1 (9454 bp), was absent from all of the BCGsubstrains currently used as TB vaccines worldwide and it was concludedthat the deletion of RD1 appeared to have occurred very early during thedevelopment of BCG, probably prior to 1921 (Behr et al., 1999). It isreasonable to hypothesize that RD1 was the primary attenuating mutationfirst isolated by Calmette and Guerin to generate BCG from M. bovis.Attempts to restore virulence to BCG with RD1-complementing clones havebeen unsuccessful (Mahairas et al., 1996).

Results

RD1 deletions of M. bovis and M. tuberculosis are attenuated forvirulence in immunocompromised mice. To test if RD1 is essential forvirulence in M. bovis and M. tuberculosis, it was necessary to deletethe RD1 (FIG. 1 a) from virulent strains, demonstrate loss of virulence,and then restore virulence by complementation with the RD1 DNA. Sincethe original M. bovis parent of BCG was lost in World War I (Grange etal., 1983), we initiated studies with virulent M. bovis Ravenel and avariety of virulent M. tuberculosis strains. Despite success ingenerating an unmarked deletion mutant of RD1 in M. tuberculosis with aplasmid transformation system^(1,2), over 100 independenttransformations failed to yield an RD1 deletion in M. bovis. As analternative strategy, specialized transduction (Bardarov et al., 2002)³was successfully used to generate RD1 deletion mutants not only in M.bovis Ravenel, but also the H37Rv, Erdman, and CDC1551 strains of M.tuberculosis (FIG. 12). This deletion represents the largest deletionmutation generated by a targeted disruption in M. tuberculosis or M.bovis made to date and demonstrates the utility of the specializedtransduction system. Moreover, since the parental specializedtransducing phage has been shown to infect over 500 clinical M.tuberculosis isolates (Jacobs et al., 1987), it should be possible tointroduce the RD1 deletion into any M. tuberculosis or M. bovis isolateof interest.

To determine if the RD1 deletion causes an attenuating phenotype in M.bovis and M. tuberculosis, the M. tuberculosis H37Rv ΔRD1 was inoculatedintravenously into immunocompromised mice possessing the SCID (severecombined immunodeficiency) mutation. Groups of ten mice were injectedintravenously with either 2×10⁶ wild type or ΔRD1 strain of M.tuberculosis and M. bovis, and three mice per group were sacrificed 24hours later to verify the inoculation doses. All of the SCID miceinfected with the parental M. tuberculosis or M. bovis strain diedwithin 14 to 16 days post-infection (FIG. 12A). In contrast, the SCIDmice infected with equal doses of the ΔRD1 strains of M. tuberculosis orM. bovis were all alive at 25 to 41 days post-infection, demonstrating ahighly significant attenuation of the virulence of both strains. It isimportant to note that BCG-Pasteur kills SCID mice approximately 70 dayspost-infection (FIG. 13B), suggesting that BCG substrains have acquiredadditional attenuating mutations which are consistent with the deletionanalysis of BCG strains (Behr et al., 1999) and the previous failures torestore virulence with the RD1 region (Mahairas et al., 1996).

To prove that the attenuation of virulence was due to the RD1 deletion,the M. tuberculosis ΔRD1 was transformed with an integrating cosmid,2F9, containing the RD1 region from M. tuberculosis H37Rv⁴. SCID micewere infected as described above and the attenuation for virulence wasrestored to the parental virulent phenotype (FIG. 13B). These resultsstrongly suggest that the genes deleted from the RD1 region contributeto the virulence phenotype.

The M. tuberculosis ΔRD1 is highly attenuated in immunocompetent BALB/cmice. The virulence of the M. tuberculosis ΔRD1 mutant was furtherassessed by intravenous inoculation of immunocompetent BALB/c mice.While the virulent parent M. tuberculosis strain killed the BALB/c micein 10 to 17 weeks post-infections, 100% of mice were alive at 48 weeksand 43 weeks post-infections in two independent experiments (FIG. 13C).

While infection with BCG and M. tuberculosis ΔRD1 yielded similarsurvival results in BALB/c mice, there were substantial differences inthe growth kinetics in mice. BCG grew in a limited fashion in lungs,liver and spleen in BALB/c mice during the 22 weeks of the experiment(FIGS. 4B-D). In contrast, the M. tuberculosis ΔRD1 strain grew in afashion indistinguishable from the parental M. tuberculosis H37Rv in allmouse organs for the first 8 weeks. Thereafter, mice infected with theparental M. tuberculosis failed to contain the infection leading tomortality. Strikingly, mice infected with the M. tuberculosis ΔRD1showed a definite control over infection resulting in significantlyprolonged survival of mice (FIGS. 4B-D).

Histopathological examination further demonstrated that the mutant wasattenuated in virulence compared to the parent strain H37Rv (FIGS.5D-F). In contrast to the rapidly progressive infection with the parentstrain, the lung lesions caused by the mutant were maximal in miceexamined at 8 weeks post-infection. Consolidating granulomatouspneumonia involved an estimated 25-30% of the lung in these mice.Numerous organisms were demonstrated by acid fast staining. Thepneumonia subsequently underwent partial resolution. By 14 weeks, andagain, at 22 weeks post-infection, the lungs showed peribronchial andperivascular inflammatory cell accumulations and focal, generallynon-confluent, granulomas now with a promiment lymphocytes infiltration.The numbers of acid fast bacilli were reduced. Liver lesions consistedof low numbers of scattered granulomas. Spleens were smaller, withpersistent granulomas in the red pulp. Mice infected with M. bovis BCGshowed mild lesions in the lung, liver and spleen at all time points(FIG. 5G-I). At longer time intervals post-infection the lesions werefewer in number, and smaller with prominent lymphocytic infiltrations.At 14 weeks post-infection, M. bovis BCG was below the level ofdetection by acid fast staining. In summary, whereas M. tuberculosisΔRD1 initially grew in a manner similar to the parental M. tuberculosisH37Rv, this RD1 mutant was limited in the extent of spread of infection,particularly in the lung. This contrasts the extensive and severe damagecaused by the parent strain. The subsequent resolving granulomas,localization of the lesions and changes in the composition of theinflammatory cell infiltrations suggested that the mice mounted aneffective immune response to combat M. tuberculosis ΔRD1 infection andthereby reduced the numbers of viable organisms.

Early BCG properties: Altered colonial morphotypes and long-termimmunogenicity. While frozen stocks of the original BCG strain do notexist, written records do exist describing the early BCG strains, as Dr.Calmette sent the strains to many laboratories. In a study published in1929, Petroff and colleagues reported that BCG displayed two distinctcolony types (Petroff et al., 1929). One morphotype was a smooth (S)phenotype that was flat and corded (like the parental virulent strain)and the second was a rough and raised (R) phenotype. The M. tuberculosisΔRD1 mutant was generated independently four times and consistentlyyielded a 20 to 50% mixture of two colonial morphotypes on Middlebrookmedium without Tween 80 (FIG. 3 b). The distinction of these two typesof morphology could be noted even when the colonies were less than twoweeks old, as the rough colonies were constricted and elevated with onlya small portion of the base of the colony attached to the agar, whilethe smooth colonies tended to be flattened and spread out. When coloniesgrew older, e.g. 6 weeks old, the rough colonies remained moreconstricted compared to those of smooth colonies. The rough coloniesexhibited large folds on the surface (FIG. 3 f-g), as compared to thoseof the smooth colonies that exhibited small wrinkles (FIG. 3 e).

The generation of two distinct colonial morphotypes must be a phenotypicchange induced by the deletion of RD1. The morphotypes could not becloned, as repeated subculturing of either the R or S colonies continuedto yield both colonial morphotypes. Moreover, Southern analysis of R andS colonies revealed each morphotype had the same RD1-deleted genotype(FIG. 3D). Furthermore, complementation of M. tuberculosis ΔRD1 with theRD1 region restored the mutant phenotype back to the homogenous parentalS phenotype (FIG. 3 a-c). These results suggest that the variablemorphotypes resulted directly from the RD1 deletion. It can therefore bepostulated that a regulator of colonial morphology is affected by one ormore of the deleted genes.

The generation of two distinct colonial morphotypes must be a phenotypicchange induced by the deletion of RD1. The morphotypes could not becloned, as repeated subculturing of either the R or S colonies continuedto yield both colonial morphotypes. Moreover, Southern analysis of R andS colonies revealed each morphotype had the same RD1-deleted genotype(FIG. 3D). Furthermore, complementation of M. tuberculosis ΔRD1 with theRD1 region restored the mutant phenotype back to the homogenous parentalS phenotype (FIG. 3A-C). These results suggest that the variablemorphotypes resulted directly from the RD1 deletion. It can therefore bepostulated that a regulator of colonial morphology is affected by one ormore of the deleted genes.

One of the hallmark characteristics of BCG is its ability to provideprotection against aerosolized challenge with virulent M. tuberculosis.To test the potential of M. tuberculosis ΔRD1 to immunize and protectmice against tuberculous challenge, we used the model of subcutaneousimmunization of C57BL/6 mice followed by an aerogenic challenge withvirulent M. tuberculosis (McGuire et al., 2002). Groups of mice werevaccinated subcutaneously with either 1×10⁶ BCG 9 or 1×10⁶ M.tuberculosis ΔRD1. Eight months following vaccination, the mice were allhealthy, thereby demonstrating attenuation in a third mouse strain.Vaccinated and unvaccinated mice were aerogenically challenged with 200CFU of the acriflavin-resistant strain of M. tuberculosis Erdman.Twenty-eight days after the challenge, the mice were sacrificed and thebacterial burden in the lungs and spleens were determined (see Table 5).Naive mice served as controls. While the acriflavin-resistant M.tuberculosis grew to 6.61±0.13 (log¹⁰ CFU) in lungs of naive mice, boththe BCG-vaccinated and M. tuberculosis ΔRD1-vaccinated mice exhibitedgreater than 1 log protection in lungs with CFU values of 5.07±0.10(p<0.001 relative to controls) and 5.11±0.14 (p<0.001), respectively,detected at the four week time point. The M. tuberculosis ΔRD1 alsoprotected against hematogenous spread; CFU values in the spleen were5.26±0.11 for the controls, 4.00±0.33 (p<0.01) for the M. tuberculosisΔRD1 immunized mice, and 3.85±0.17(p<0.01) for the BCG vaccinatedanimals. Thus, the M. tuberculosis ΔRD1 shares long-term immunogenicitylike BCG.

TABLE 5 Bacterial burden of virulent M. tuberculosis in uninoculatedmice and mice inoculated with BCG and H37Rv ΔRD1. Vaccination strainLung (log₁₀CFU) Spleen (log10CFU) — 6.61 ± 0.13 5.26 ± 0.11 BCG 5.07 ±0.10*** 3.85 ± 0.17** H37Rv ΔRD1 5.11 ± 0.14*** 4.00 ± 0.33** **p <0.01; ***p < 0.001.Discussion

BCG is a mutant of M. bovis that was isolated over 94 years ago andcharacterized for its attenuation for virulence in animals. For over 80years, BCG has been used as a tuberculosis vaccine having been given to3 billion humans. It is currently the only anti-tuberculous vaccineavailable for use in humans, yet its precise attenuating mutations andmechanisms of attenuation have never been determined. Previous studieshad identified regions of the M. bovis chromosome that were absent fromBCG, but present in virulent M. bovis and M. tuberculosis strains(Mahairas et al., 1996; Gordon et al., 2001). An elegant microarrayanalysis has also demonstrated that there was only one deletion commonto all BCG strains; the authors hypothesized this was the primaryattenuating mutation in the original BCG strain isolated by Drs.Calmette and Guerin (Behr et al., 1999).

Using a combination of targeted deletion mutagenesis, virulence assays,and complementation analysis, we have been able to unambiguously provethat RD1 is required for virulence for M. tuberculosis, and by analogyfor M. bovis, for the first time.

Moreover, the combination of phenotypes associated with the early BCGstrains: i) the attenuation for virulence, ii) the altered colonialmorphotypes, and iii) the ability to confer long-term immunogenicity inanimals allow us to conclude that the RD1 deletion was the primaryattenuating mutation in the original BCG isolate.

With regards to the ΔRD1 mutant histology, at 22 weeks post infection,it was noted that the mutant was limited in the extent of the spread ofinfection, in contrast to the extensive damage caused by the parentalstrain. Interestingly, Pethe et al. (2001) determined that M.tuberculosis needs to bind and/or invade epithelial cells in order todisseminate and cause widespread destruction of the lung, whilst anotherstudy reported that pulmonary M cells can act as a portal of entry tothe lung for the tubercle bacilli (Teitelbaum, 1999). In relation to invitro analyses, studies utilizing a model of the alveolar barrier,consisting of pneumocytes and monocytes, described how M. tuberculosisinfection of the pneumocytes resulted in cytolysis, which disrupted thebarrier and allowed more efficient translocation of intracellularbacilli (Bermudez et al., 2002).

Notes

¹The following four primers were used to amplify upstream and downstreamflanking sequences (UFS and DFS, respectively) for the construction ofthe RD1 deletion mutants. UFS was amplified using TH201:GGGGGCGCACCTCAAACC (SEQ ID NO:5) and TH202: ATGTGCCAATCGTCGACCAGAA (SEQID NO:6). DFS was amplified using TH203: CACCCAGCCGCCCGGAT (SEQ IDNO:7), and TH204: TTCCTGATGCCGCCGTCTGA (SEQ ID NO:8). Recognitionsequences for different restriction enzymes were included at the ends ofeach primer to enable easier manipulation.

²The unmarked deletion mutant of M. tuberculosis H37Rv, mc²4002, wasgenerated by transformation using a sacB counterselection (Snapper etal., 1988; Pelicic et al., 1996; Pavelka et al., 1999). Specifically,the plasmid pJH508 was created by first cloning UFS into KpnI and XbaIsites, then cloning DFS into EcoRI and HindIII sites of pJH12, apMV261-derived E. coli—Mycobacteria shuttle plasmid, to create pJH506 inwhich UFS and DFS flanked a green fluorescent protein gene (GFPuv,Clonetech) whose expression was driven by the M. leprae 18Kd promoter.The UFS-gfp-DFS cassette was sub-cloned into the EcoRV site of plasmidpYUB657 to create pJH508. The first homologous recombination involvedthe identification of hygromycin resistant colonies, resulting from thetransformation of M. tuberculosis with pJH508. Southern analysis of theNcoI-digested DNA isolated from hygromycin resistant colonies probedwith UFS or DFS, confirmed the presence of a single copy of pJH508inserted into the M. tuberculosis genome. The transformant (mc²4000)identified was then grown in 7H9 broth to saturation, to allow thesecond homologous recombination to occur, resulting in recombinants thatcould be selected by plating the culture on 7H10 plates, supplementedwith 3% sucrose. Both Southern analysis and PCR of the DNA isolated fromsucrose resistant colonies confirmed the RD1 deletion.

³Specialized transduction is a mycobacteriophage-based method for thedelivery of homologous DNA constructs using conditionally replicatingshuttle phasmids (Jacobs et al., 1987; Bardarov et al., 1997; Carriereet al., 1997) has been used successfully for M. tuberculosis (Glickmanet al., 2000, 2001; Raman et al., 2001). Specifically, a transducingphage phAEKO1 was constructed by inserting UFS and DFS into pJSC347,flanking a hygromycin cassette, to create pJH313. pJH313 was digestedwith PacI and ligated to phAE159, a temperature-sensitivemycobacteriophage derived from TM4. The transduction was performed bygrowing M. tuberculosis to an O.D.₆₀₀ of 1.0, washing twice with MPbuffer (50 mM Tris pH 7.6, 150 mM NaCl, 10 mM MgCL₂, 2 mM CaCl₂),resuspending into an equal volume of MP buffer and mixing with thetransducing phage phAEKO1 at an MOI of 10. The mixtures were incubatedat 37° C. overnight, then plated on 7H10 plates supplemented withhygromycin at 50 μg/ml. Hygromycin resistant colonies were analyzed byPCR and Southern analysis, as described above, to confirm the deletionof RD1.

⁴Complementation analyses was performed using the integration proficientcosmids (Skjot et al., 2000; van Pinxteren et al., 2000) pYUB412 made byS. Bardarov, a library made by F. Bange, and cosmid identified andgenerously provided by S. T. Cole.

EXAMPLE 5 Vaccine Efficacy of a Lysine Auxotroph of M. Tuberculosis

In this Example, we describe the in vivo growth phenotype and vaccineefficacy of a lysine auxotrophic mutant of Mycobacterium tuberculosisstrain H37Rv. An immunization experiment using the mouse model with anaerosol challenge showed that two doses of the M. tuberculosis mutantwere required to generate protection equivalent to that of the BCGvaccine.

Despite the existence of anti-microbial drugs and a widely used vaccine,Mycobacterium tuberculosis remains the primary cause of adult death dueto a bacterial agent (Dolin et al., 1994). The emergence of multi-drugresistant strains of M. tuberculosis, the variable efficacy of thecurrent vaccine, the bacille-Calmette and Geurin (BCG), and the HIVpandemic have all contributed to a growing global tuberculosis problem.

Several studies have described the development of attenuated auxotrophicstrains of BCG and/or M. tuberculosis (Guleria et al., 1996); Hondaluset al., 2000; Jackson et al., 1999; Smith et al., 2001). All of thesestudies utilized single immunization protocols and demonstrateddifferences in the protective responses thus elicited. In this study, wedescribe the in vivo growth characteristics of a previously describedlysine auxotroph of M. tuberculosis H37Rv (Pavelka and Jacobs, 1999),and evaluate the vaccine potential of this mutant by a multipleimmunization protocol in a mouse model of the human disease, using anaerosol challenge.

Clearance of the M. tuberculosis lysine auxotroph in SCID mice. FemaleSCID mice were bred at the animal facility of the Albert EinsteinCollege of Medicine. The animals were maintained under barrierconditions and fed sterilized commercial mouse chow and water adlibitum. The M. tuberculosis strains Erdman, mc²3026 (ΔlysA::res) (Id.),and mc²3026 bearing pYUB651 (expressing the wild-type lysA gene) weregrown in Middlebrook 7H9 broth (Difco) supplemented with 0.05% Tween-80,0.2% glycerol, 1×ADS (0.5% bovine serum albumin, fraction V (Roche);0.2% dextrose; and 0.85% NaCl) or on Middlebrook 7H10 or 7H11 solidmedium (Difco) supplemented with 0.2% glycerol and 10% OADC (BectonDickinson). Culture media for the lysine auxotroph were supplementedwith 1 mg/ml of L-lysine (for both liquid and solid media), and 0.05%Tween-80 was also added to solid medium. Liquid cultures were grown in490 cm² roller bottles (Corning) at 4-6 rpm. Plates were incubated for3-6 weeks in plate cans. All cultures were incubated at 37° C.

Titered frozen stocks of the bacteria were thawed and dilutedappropriately in phosphate buffered saline containing 0.05% Tween-80(PBST). The bacterial suspensions were plated at the time of injectionto confirm the number of viable bacteria. Intravenous injections weregiven via a lateral tail vein. At various time points post-injection (24hours, then once weekly), 3 mice were sacrificed, and the lungs, liver,and spleen removed and homogenized separately in PBST using a Stomacher80 (Tekmar, Cincinnati, Ohio). The homogenates were diluted in PBST andplated to determine the number of CFU/organ. Note that mice weresacrificed at 24 hours post-injection in order to compare the bacterialcolony forming units recovered from the mice with the colony formingunits in the suspensions at the time of injection. Thus the bacterialcounts reported at time zero actually represent the viable bacteriarecovered from the mice at 24-hours post-injection.

The lysine auxotrophic strain was cleared from and did not appear togrow in the examined organs of the SCID mice, while the complementedstrain multiplied extensively (FIG. 14). Interestingly, the auxotrophicinoculum was cleared from the spleens and lungs but persisted somewhatlonger in the liver (FIG. 14B). The mice receiving the complemented M.tuberculosis mutant died within three weeks of challenge, while the micegiven the auxotrophic M. tuberculosis mutant did not display any grossorgan pathology and survived for at least the duration of theexperiment.

Two immunizations with the M. tuberculosis lysine auxotroph mc²3026 arerequired to match the efficacy of vaccination with BCG-Pasteur. Wetested the vaccine potential of the lysine auxotroph mc²3026 in themouse model by means of a virulent aerosol challenge. Female,pathogen-free C57BL/6 mice (Jackson Laboratories, Bar Harbor, Me.) werevaccinated intravenously with ca. 1×10⁶ CFU of the M. tuberculosislysine auxotroph or BCG-Pasteur suspended in 0.2 ml PBST. Micevaccinated with mc²3026 were revaccinated at 4 week intervals and thenumber of viable organisms in the lungs and spleens determined weeklythroughout the vaccination period, as described above for the SCID mouseexperiments. Five mice were examined at each time point.

Immunized mice were challenged 3 months after the initial vaccination. Afrozen aliquot of a M. tuberculosis Erdman stock was thawed and dilutedin PBST to ca. 1×10⁶ CFU/ml and 10 ml was introduced into the nebulizerof a Middlebrook aerosol chamber (Glas-Col, Terre Haute, Ind.). The micewere exposed to the infectious aerosol for 30 minutes, inhaling 50-100CFU into their lungs over this period. Five mice were sacrificedimmediately following the challenge period and the lung homogenates wereplated to check the amount of the challenge inoculum actually reachingthe lungs. Groups of vaccinated and control mice were sacrificed 14, 28,and 42 days later and the lung and spleen homogenates plated todetermine the number of viable colony forming units of M. tuberculosisErdman present. Data were analyzed using the Student's t-test and ananalysis of variance between several independent means, using the InStat Statistics program (GraphPad Software, San Diego).

A preliminary experiment demonstrated that a single intravenousimmunization of immunocompetent C57BL/6 mice with the M. tuberculosismutant did not generate a significant protective response to thesubsequent aerosol challenge with virulent M. tuberculosis Erdman. Inthat experiment, the M. tuberculosis auxotroph was rapidly cleared fromthe mice (FIG. 15A), and the single immunization with the auxotroph wasinsufficient to reduce the bacterial burden in the lungs and spleensrelative to a single immunization with BCG (FIG. 15B).

The failure of the auxotroph to confer protection might have been due tothe inability of the mutant to persist long enough, or to synthesizeenough antigen to induce an immune response that could significantlyrestrict the growth of the challenge organisms. One way to circumventthis problem is to give multiple doses of vaccine (Collins, 1991;Homchampa et al., 1992). To this end, mice were intravenously immunizedtwo or three times at four-week intervals with the M. tuberculosislysine auxotroph. In both cases, the vaccine strain was cleared from thelungs and spleens of all the mice at rates similar to that seen with thesingle immunization experiment (FIG. 15A). Three months after the firstimmunization the mice were challenged with M. tuberculosis Erdman by theaerosol route and the bacterial counts in the lungs and spleens weredetermined and compared to a BCG-Pasteur immunized control, as well asthe sham immunized controls. As seen in FIG. 15C, double immunizationwith the M. tuberculosis lysine auxotroph induced a protective responsethat was equivalent to that of the BCG control. The reduction in countsin the lung and spleen was equivalent to a 100-fold reduction inbacterial counts compared to the unvaccinated control (FIG. 15C). Theresults from the triple immunization experiment were essentially similaras those from the double immunization experiment described above (datanot shown). Furthermore, mice that were immunized with three doses ofthe M. tuberculosis lysine auxotroph and challenged with virulent M.tuberculosis Erdman survived at least as long as the BCG-immunizedcontrol mice (FIG. 16).

Several studies have described the development and vaccine efficacy ofattenuated mutant strains of M. tuberculosis (Jackson et al., 1999;Hondalus et al., 2000; Smith et al., 2001). The first study reportedthat a purine auxotroph of M. tuberculosis was unable to grow inmacrophages and was attenuated for growth in both mice and guinea pigs(Jackson et al., 1999). A guinea pig vaccination experiment determinedthat a single immunization with the auxotroph allowed the animals torestrict the growth of virulent M. tuberculosis in the lungs as well asa single immunization with wild-type BCG, following aerosol challenge.However, the reduction in growth of the challenge organism in the spleenafforded by the auxotroph was not as extensive as that afforded by BCG.Another study reported that a leucine auxotroph of M. tuberculois Erdmancannot grow in macrophages and is avirulent to immunocompromised SCIDmice (Hondalus et al., 2000). Immunocompetent mice vaccinated once witha M. tuberculosis leucine mutant did not significantly restrict thegrowth of the virulent challenge organism in the lungs or spleen as muchas the control mice vaccinated with BCG (Id.). However, the miceimmunized with the leucine auxotroph survived as long as the BCGimmunized controls and exhibited a decreased histopathology relative tothat seen in the non-immunized controls (Id.). A third study showed thatM. tuberculosis proline and tryptophan auxotrophs were attenuated and asingle immunization of mice with either of these mutants affordedprotection against an intravenous challenge with virulent M.tuberculosis, comparable to that for BCG, as indicated by the meansurvival times (Smith et al., 2001). In those experiments, miceimmunized with pro or trp mutants could restrict the growth of thechallenge organisms to the same extent as mice immunized with BCG,although the magnitude of protection in either case (M. tuberculosisauxotrophs or BCG) was not as extensive as that seen in the otherstudies (Id.).

In the present study we have demonstrated that a single immunization ofmice with the avirulent M. tuberculosis lysine auxotroph did notgenerate an immune response capable of significantly restricting thegrowth of virulent M. tuberculosis Erdman following an aerogenicchallenge. However, administration of a second or a third dose of thisvaccine increased protection substantially, as measured by the number ofviable bacteria per organ, to a level similar to that achieved withsingle dose of BCG-Pasteur. This level of protection did not seem to begreatly increased by a third dose of vaccine, although the triplyimmunized mice survived as long as the control mice immunized with asingle dose of BCG-Pasteur. Mice that were immunized twice were notfollowed to determine mean survival time, but comparing the growthcurves of the challenge bacteria following the double and tripleimmunizations, it seems likely that the survival time for the doublyimmunized mice would be much the same as that for the triple-immunizedmice.

The previous studies using M. tuberculosis auxotrophs as vaccine strainsshowed substantial variations in their effectiveness. This variabilityis likely to be due to a number of factors, including the different M.tuberculosis background strains used to construct the mutants, differentmouse strains used in the various protection studies, and the differentchallenge organisms and challenge routes used. There was alsoconsiderable variation in the protective efficacy of the differentvaccines compared to that observed in controls using BCG immunization.These differences pose a number of questions concerning the bestindicators of protection, especially in the long term. Should viablebacterial counts or survival be the primary indicator of protection orshould both be given equal weight? The results of this study indicatethat more than one immunization with a M. tuberculosis lysine auxotrophdid generate a significant protective response as indicated by bothcriteria. We believe it is important that multiple immunizationprotocols be considered in the further development of attenuated M.tuberculosis strains as potential human vaccines.

This is the first study demonstrating that a multiple immunizationprotocol using an auxotroph of M. tuberculosis can protect against ahighly virulent aerosol challenge compared to that seen for BCG. SinceBCG vaccines have shown variable efficacy when tested in humans, anauxotrophic M. tuberculosis vaccine might represent an attractivebooster vaccine with which to augment childhood BCG immunization.

EXAMPLE 6 Mutants of Mycobacterium Tuberculosis HAving Two AttenuatingMutations are Safe and Provide Protection in Mammals Against Challengefrom Virulent Mycobacteria

The experiments described in this Example employ materials and methodsdescribed in the other Examples.

Construction and characterization of M. tuberculosis ΔRD1 ΔpanCD(mc²6030). A pantothenate auxotroph of M. tuberculosis ΔRD1 wasgenerated by specialized transduction and the strain designated mc²6030.No CFU were detected on 7H11 when 5×10¹⁰ CFU were plated (repeatedtwice), suggesting the reversion frequency to be below 10⁻¹¹.

SCID mice infected with 1×10² CFU H37Rv succumbed to infection in 6weeks, whereas the mice infected with 1×10⁶ mc²6030 survivedsignificantly longer with more than 75% of mice surviving for more than300 days (FIG. 17A). Bacteria isolated from mc²6030-infected mice beforethey died were all auxotrophs, confirming that there were no revertantsunder in vivo conditions. In order to assess the safety of mc²6030 inimmunocompetent BALB/c mice, we infected mice intravenously with 1×10⁶mc²6030 or 1×10⁶ of wild-type H37Rv. All mice infected with H37Rvsuccumbed to infection by 150 days, whereas mice infected with mc²6030survived for more than 300 days (FIG. 17B). In an effort to understandthe role of immune responses in controlling infection with thepantothenate mutants, we infected immunocompetent C57B1/6 with 1×10⁶ CFUof mc²6001 (ΔRD1), mc²6004 (complementing strain), mc²6030 (ΔRD1 ΔpanCD)or wild-type H37Rv. Mice infected with H37Rv and mc²6004 showedprogressive growth in all the three organs, whereas mice infected withmc²6030 showed a drop in growth during the first 3 weeks in the lungsand spleen (FIG. 18). Following 3 weeks of infection, the growth patternof both mc²6001 and mc²6030 were identical in the spleen and lungs. Miceimmunized subcutaneously with one or two doses of mc²6030 demonstratedprotection against aerosol challenge with virulent M. tuberculosis,which was comparable to the protection afforded by BCG vaccination(Table 6). No pantothenate auxotrophs were recovered from spleen orlungs of mice at 1, 2 or 3 months following subcutaneous immunization.

TABLE 6 Bacterial burden of virulent M. tuberculosis in uninoculatedmice and mice inoculated with BCG or one or two doses of ΔRD1ΔpanCD.Experimental Group Lung CFUs (log₁₀) Spleen CFUs (log10) Naive 5.99 ±0.09 4.94 ± 0.06 ΔRD1ΔpanCD (1 dose) sc 5.22 ± 0.10* 4.04 ± 0.15*ΔRD1ΔpanCD (2 doses) sc 4.86 ± 0.14** 3.58 ± 0.11** BCG (1 dose) sc 4.79± 0.19** 3.73 ± 0.27** *p < 0.01 relative to controls; **p < 0.001relative to controls

Construction and characterization of M. tuberculosis ΔlysAΔpanCD(mc²6020). A pantothenate auxotroph of M. tuberculosis ΔlysA wasgenerated by specialized transduction and the strain designated mc²6020.No CFU were detected on 7H11 when 5×10¹⁰ CFU were plated, suggesting thereversion frequency to be below 10⁻¹¹. This double mutant is auxotrophicfor both lysine and pantothenate. SCID mice infected with 1×10² CFUH37Rv succumbed to infection in 6 weeks, whereas the mice infected with1×10⁶ mc²6020 survived for more than 400 days with no mortality. Inorder to assess the safety and growth kinetics of mc²6020 inimmunocompetent BALB/c mice, we infected mice intravenously with 1×10⁶mc²6020 or 1×10⁶ of wild-type H37Rv. All mice infected with H37Rvsuccumbed to infection by 150 days, whereas mice infected with mc²6020survived for more than 400 days. After 3 weeks following intravenousinfection, no colonies of mc²6020 could be recovered from spleen, liveror lungs of infected mice. Interestingly, mice immunized subcutaneouslywith one or two doses of mc²6020 demonstrated protection against aerosolchallenge with virulent M. tuberculosis, which was comparable to theprotection afforded by BCG vaccination (Table 7). No pantothenate andlysine requiring auxotrophs were recovered from spleen or lungs of miceat 1, 2 or 3 months following subcutaneous immunization. Other studiesestablished that both mc²6020 and mc²6030 protects the a level ofprotection of mice against TB equivalent to the protection afforded byBCG (FIG. 19).

TABLE 7 Bacterial burden of virulent M. tuberculosis in uninoculatedmice and mice inoculated with BCG or one or two doses of mc²6020(ΔlysAΔpanCD) sc or one dose of mc²6020 iv. Experimental group Lung CFUs(log₁₀) Spleen CFUs (log₁₀) naive 6.03 ± 0.05^(a) 4.84 ± 0.27 BCG (1dose) sc 4.76 ± 0.19*** 3.95 ± 0.18* mc²6020 (1 dose) sc 5.05 ± 0.06***4.02 ± 0.11* mc²6020 (2 doses) sc 5.09 ± 0.05*** 4.06 ± 0.27 mc²6020 (1dose) iv 5.06 ± 0.11*** 4.00 ± 0.15* ^(a)Mean ± SEM p < 0.001 =***; p <0.05 =*

These data clearly demonstrate the safety and immunogenicity of thesetwo double mutants of M. tuberculosis in mice.

The double deletion mutant mc²6030 (ΔRD1ΔpanCD) immunizes and protectsSCID mice from aerosolized M. tuberculosis challenge. The doubledeletion mutants were safer than BCG in SCID mice, where all of the SCIDmice died before 100 days when inoculated with BCG, 100% and 25% of themice survived inoculation with mc²6020 and mc²6030, respectively (FIG.20).

In view of the above, it will be seen that the several advantages of theinvention are achieved and other advantages attained.

As various changes could be made in the above methods and compositionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description and shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

All references cited in this specification are hereby incorporated byreference. The discussion of the references herein is intended merely tosummarize the assertions made by the authors and no admission is madethat any reference constitutes prior art. Applicants reserve the rightto challenge the accuracy and pertinence of the cited references.

1. A mycobacterium in the Mycobacterium tuberculosis complex,genetically engineered to be auxotrophic for a vitamin.
 2. Themycobacterium of claim 1, wherein the mycobacterium is a Mycobacteriumbovis.
 3. The mycobacterium of claim 1, wherein the mycobacterium is aMycobacterium tuberculosis.
 4. The mycobacterium of claim 3, wherein theM. tuberculosis exhibits attenuated virulence in a mammal when comparedto the M. tuberculosis without the deletion.
 5. The mycobacterium ofclaim 3, further comprising a foreign DNA stably integrated into genomicDNA of the M. tuberculosis.
 6. The mycobacterium of claim 1, wherein thevitamin is pantothenic acid.
 7. The mycobacterium of claim 6, whereinthe deletion is a ΔpanCD deletion.
 8. The mycobacterium of claim 1, 2,3, 4, 6 or 7, further comprising a deletion controlling production of anamino acid.
 9. The mycobacterium of claim 8, wherein the amino acid islysine.
 10. A non-naturally occurring Mycobacterium tuberculosiscomprising a deletion of the entire RD1 region, wherein the M.tuberculosis with the RD1 deletion exhibits attenuated virulence in amammal when compared to virulent M. tuberculosis without the deletion.11. The M. tuberculosis of claim 10, which is genetically engineered.12. The M. tuberculosis of claim 10, wherein the mammal isimmunocompromised.
 13. The M. tuberculosis of claim 10, wherein the RD1region has at least 95% homology to SEQ ID NO:1.
 14. The M. tuberculosisof claim 10, further comprising a second deletion.
 15. The M.tuberculosis of claim 14, wherein the second deletion causes the M.tuberculosis to be auxotrophic.
 16. The M. tuberculosis of claim 15,wherein the second deletion is a region controlling production of avitamin.
 17. The M. tuberculosis of claim 16, wherein the vitamin ispantothenic acid.
 18. The M. tuberculosis of claim 17, wherein thesecond deletion is a ΔpanCD deletion.
 19. The M. tuberculosis of claim15, wherein the second deletion is in a region controlling production ofan amino acid.
 20. The M. tuberculosis of claim 19, wherein the aminoacid is lysine.
 21. The M. tuberculosis of claim 19, wherein the seconddeletion is a ΔlysA deletion.
 22. The M. tuberculosis of claim 10,further comprising a foreign DNA stably integrated into genomic DNA ofthe M. tuberculosis.
 23. The M. tuberculosis of claim 22, wherein theforeign DNA encodes at least one protein or polypeptide selected fromthe group consisting of an antigen, an enzyme, a lymphokine, animmunopotentiator, and a reporter molecule.